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MOLECULAR BIOLOGY Translation Kolluru. V. A. Ramaiah Professor Department of Biochemistry University of Hyderabad (Revised 30-Oct-2007) CONTENTS Introduction Messenger RNA (mRNA) Splicing Addition of 5’Cap Addition of poly A tail RNA editing Ribosomes Subunits, composition, morphology Processing of rRNA Functions of ribosomal subunits Polysomes Transfer RNA (tRNA) Processing Modified bases Charging or aminoacylation Genetic Code Cell-free translational systems Synthetic mRNA templates Amino acid analyses of polypeptides produced by synthetic templates Codon- charged tRNA- ribosome complexes Wobble and degeneracy Mutations Gene density and overlapping genes Protein synthesis Initiation Elongation Termination Ribosome recycling Differences in the initiation in eukaryotes and prokaryotes Inhibitors Translational regulation Different RNAs and functions Co- and Post-translational Modifications of proteins Keywords Messenger RNA (mRNA); Ribosomes; Polysomes; Transfer RNA (tRNA); Aminoacylation; Genetic code; Translational system; Wobble; Mutations; Gene density; Protein synthesis; Translational regulation. 2 Introduction Proteins are biological polymers like the nucleic acids. The alphabets of protein language are however different from that of nucleic acids. The monomeric units of proteins are the alphaamino acids. The twenty naturally occurring amino acids are like the alphabets of a language that go into the composition of different proteins. A typical α-amino acid consists of an amino group (-NH2), carboxyl group (-COOH) and an R group. It is the R group that gives specificity to an amino acid. The carboxyl group is attached to the α-carbon, the carbon next to the carboxyl group (Fig. 1). Amino acids in a protein chain are linked by a peptide bond ( ). Usually, proteins have an N-terminal end carrying a free amino group and C-terminus with –COOH. The linear sequence of amino acids in each protein is specific like the alphabets in a word. Proteins are vital for life and mediate a variety of functions: in storage, structure, catalysis, signaling, transport, defense, transporting ions, blood clotting, and muscle contraction (Fig.2). Proteins like prions even mediate transmissible diseases called spongiform encephalopathies and the structure of a protein determines its function. Hence the study of biological synthesis of proteins and their modifications to attain a proper 3-dimensional structure are important. Approximately 35-45% genes make products devoted for translation, and 35-40% of the total energy generated, for example by E. coli, is consumed for protein synthesis. Hence the synthetic process and also the modification process require close monitoring and regulation. The information for the synthesis of a protein is stored in the nucleotide sequence of messenger RNA (mRNA) that in turn is synthesized from the corresponding DNA. The process of RNA synthesis from a DNA template is called transcription and the synthesis of proteins from an mRNA template is called translation. The information flow also occurs from an RNA template to a DNA molecule through a process called reverse transcription where DNA is synthesized from an RNA transcript in the presence of an enzyme called reverse transcriptase (Fig. 3). In prokaryotes that lack defined organelles or nucleus, the process of transcription and translation are coupled whereas in eukaryotes, the RNA synthesis occurs in the nucleus and the protein synthesis takes place in the cytosol, organelles, or on the surface of endoplasmic reticulum (ER). Organelle protein synthesis resembles prokaryotic protein synthesis. Proteins made on the surface of ER membrane are called secretory proteins (eg: serum albumin, immunoglobulins, digestive enzymes, egg white proteins etc.,) that are destined to reach various locations/organelles in the cell. Secretory/membrane proteins carry an N-terminal signal sequence that is rich in hydrophobic amino acids. Such a signal sequence is absent in cytosolic proteins. The rate of translation of eukaryotic mRNAs is slower than in prokaryotes because of 3 the spatial and temporal separation of transcription and translational events, processing of the premessenger RNAs (mRNAs) that remove the non-coding regions, and their ability to undergo distinct posttranscriptional modifications at the 5’ and 3’ends. Several complex regulatory mechanisms control the gene expression in eukaryotes and they occur at different levels: transcription, post transcription, transport of RNAs into the cytosol, translation, and the degradation or stability of mRNA. Like the synthesis of DNA (replication) and RNA (transcription), specific cellular machinery directs protein synthesis (translation). While differences exist, one can also see some common pattern directing the biological synthesis of these polymers viz., proteins, RNA and DNA. The syntheses of all these molecules require a template strand that contains the necessary information with ‘start’ and ‘stop’ signals. The addition of monomers occurs on the template. These monomers come one after another based on the sequence information coded in the template and attach to the template molecule. A covalent bond like phosphodiester bond joins the adjacent nucleotides in DNA and RNA synthesis, whereas a peptide bond brings together adjacent amino acids in protein synthesis. The primary products, or the newly made RNA and protein molecules are processed where certain nucleotides or amino acids that are non-coding or non-essential for function or structure are selectively removed to yield active molecules. Processing of RNAs and pre-proproteins yield biologically active RNA and protein molecules that are devoid of ‘introns’ as in RNA, or prepro-amino acid sequences or ‘inteins’ as in proteins. In addition, eukaryotic RNAs and the amino acids in proteins are also modified in several other ways during or after their synthesis and these are called post-transcriptional or post-translational modifications respectively that play important role in their functions (see latter). The expression of genes i.e., the synthesis of RNA and proteins is regulated or controlled by a wide variety of ways. While the DNA of a genome is replicated completely preceding the cell division but only portions of DNA are transcribed and or translated at different periods in the development or in different cell types. The genetic content between men and mice is 97.5% similar and the 2.5% difference separates mice from men. Among organisms, like human beings, the difference in the genetic content is 0.1% and that small difference distinguishes one individual from the other for their appearance, behavior and predisposition to certain diseases. Although different cell types like muscle cells, red blood cells and cells of pancreas differ in their functions, their genetic content is 100% similar because they are all derived from the same parent cell. Similarly a normal cell differs from an abnormal, or aged, stressed, diseased or virusinfected cell. An unfertilized egg differs from a fertilized egg. A dormant seed is different from germinating seed. This is because the gene expression or the types of RNAs and proteins made by these cells are different. Hence the gene expression critically regulates the development, differentiation of cell types and plays a role in health and disease. 4 In brief, the protein synthesis machinery consists of different types of RNAs (Box-1), ribosomes, enzymes such as aminoacyl synthetases that catalyze the transfer of amino acids to corresponding tRNAs, and a variety of protein factors that are required during the substeps in initiation, elongation, termination of translation and in ribosome recycling. The process of protein synthesis consumes a lot of ATP and GTP. While ATP hydrolysis is used in driving the protein / peptide synthesis and in unwinding any secondary structures in mRNA, GTP hydrolysis facilitates conformational changes in ribosomes that are required in the joining of small subunit to the large ribosomal subunit, the translocation/movement of tRNA from one codon to another and also in the detachment of the factors from ribosomes. Ribosomes, and other components of protein synthesis that are involved in the decoding of information in mRNA are relatively stable compared to the template mRNA. In addition to the three RNAs mentioned in Box-1, several other RNAs with a variety of functions play a role in the synthesis of DNA, RNA and proteins and also in controlling gene expression (see latter). Messsenger RNA (mRNA) One of the DNA strands serves as a template for the synthesis of messenger RNA (mRNA). Messenger RNAs are protein-coding RNAs containing typically three regions: a 5’UTR (untranslated region), a protein coding sequence (open reading frame or ORF) and 3’UTR (Fig. 4B). The protein coding sequence of mRNA has a ‘start’ site at the 5’side (ribosome binding site or RBS and is about 7-10 nucleotides in length) and ‘stop’ site at the 3’end. Eukaryotic mRNAs are mostly monocistronic coding for a single polypeptide whereas prokaryotic mRNAs are polycistronic with multiple starts and stop signals and coding for more than one protein. In prokaryotes that lack a defined nucleus, the process of RNA and protein syntheses are coupled. This means, as and when the mRNA is being transcribed from a DNA template, the RNA transcript is translated by ribosomes to the corresponding protein. Further, prokaryotic mRNAs lack additional features such as a cap structure at the 5’ end and poly A tail at the 3’end which are found in most eukaryotic mRNAs. In eukaryotes, the transcription or the synthesis of mRNAs occurs in the nucleus. The pre-mRNAs are called heterogenous nuclear RNA (hnRNA). The hnmRNA or pre-mRNA molecule is bound by a spliceosome that contains several proteins and uracil-rich small nuclear RNA molecules. The primary transcripts of eukaryotes are 5 unusually long and contain coding and non-coding sequences called exons and introns respectively. Because of this reason, the eukaryotic genes are called split genes. This can be demonstrated by imperfect hybridization of the DNA template with its mature or processed mRNA transcript. Part of the regions in DNA (corresponding to introns) cannot be base paired with its mature mRNA and form single stranded loops. This can be detected using an electron micrscope or by using S1 nuclease that attacks preferentially the single stranded unpaired region in DNA. The fragments of DNA generated by S1 nuclease digestion can be separated on an acrylamide/ agarose gel electrophoresis. Thus the mature RNAs do not have certain sequences that are complimentary to their template DNA molecules and these are called introns or noncoding sequences. Splicing Splicing or joining of the exons (Fig. 4A) and removal of the introns involve two successive transestrification reactions in which the phosphodiester linkages within the mRNA are broken and reformed as shown below (see figure). Introns are incidentally ancient and are found in bacterial tRNAs, but not in their mRNAs. Archaebacteria too have introns both in their tRNAs and in the ribosomal RNAs. Eukaryotes have introns in many of their pre-RNAs including mRNAs. This suggests that probably they are lost in bacterial mRNAs due to evolutionary constraints. An analysis of eukaryotic DNA sequences at the boundaries of exons and introns revealed that introns mostly have GU sequence at the beginning (on their 5’ side) and AG sequence at the end (on the 3’ side). In addition to these sequences, splicing of introns requires a pyrimidine rich sequence called the polypyrimidine tract (U/C)11 preceding the AG nucleotides at the 3’ side of intron and a branch point sequence consisting of 5’- YNYYRAY (Y represents any pyrimidine, N refers to any nucleotide and R means a purine A or G, and A is an adenine) that is present 10-40 residues upstream (that is towards the 5’end of the intron) to the above polypyrimidine tract. Fig. 4 A and B Based on the mechanism of splicing, three groups of introns are identified. Removal of group I introns, for example present in rRNAs, can proceed in the absence of any proteins. However, the splicing reaction of group I introns require the presence of guanosine and Mg2+. The concept of 6 RNA enzymes has come from such splicing reactions. The RNA enzymes like the protein enzymes increase the rate of reaction by several fold and require typical 3-D structure of their substrates. However unlike most protein enzymes, RNA enzymes act on themselves (selfsplicing) and are modified at the end of reaction. Essentially RNA enzymes have nuclease and polymerase activities. They can remove nucleotides and can also add nucleotides through a transesterification reaction. The removal of group II and III introns (premRNAs) are similar to each other except that the removal of group III introns requires the participation of a spliceosome, a complex of several RNAs and proteins. Unlike group I introns, the splicing of group II and III introns does not require guanosine. In both cases, the OH-group of adenine nucleotide that is part of the intron branch point sequence attacks the 3’end of the exon present at the 5’end of the mRNA. Afterwards, the free 5’end of intron joins the adenine nucleotide in intron to give a lariat structure, whereas the -OH group that is generated at the 3’end of exon will attack the 3’end of intron thereby releasing the intron. At the same time, two exons present at the 5’ and 3’ends of the intron are joined with each other (Fig. 4C). Alternative Splicing Alternative Splicing produces multiple mRNAs that code for different proteins where as normal splicing includes all exons and elimination of introns. The processing of pre-mRNAs is not always uniform. Some times the processing facilitates the joining of different combinations of exons of an mRNA. Also, the cleavage and polyadenylation of pre-mRNAs (Fig. 6) at different sites in the 3’ end would produce different lengths of mRNAs. For example, splicing and 3’cleavage sites of the calcitonin mRNA that produces calcitonin hormone in the cells of thyroid gland and brain differ to produce different proteins. The mature transcript of the calcitonin mRNA is found long in brain cells than in thyroid cells. This is because the thyroid calcitonin mRNA contains four of the six exons (1-4) without any introns. However in brain cells, the cleavage and polyadenylation of pre-mRNA occurs at the end of sixth exon. The processed or mature mRNA in brain cells has exons 1, 2, 3, 5 and 6 and does not have any of the introns. Exon 4 is not included. Alternative splicing may explain the diversity of proteins produced in eukaryotes that apparently do not correspond to their estimated number of functional genes. In humans, for example, approximately 30,000-35,000 functional genes code for millions of proteins. The processing of heavy chain pre-mRNAs of IgM class immunoglobulins of B cells and plasma cells, and the α-tropomysin gene in different muscle cells are some of the other examples of alternative splicing. This may be possible because of the presence /absence of specific cellular splicing factors. The plasma cell that secretes immunoglobulins into the blood produces a mature IgM transcript that retains one of the exons which produces a stretch of hydrophilic amino acids and is consistent with the ability of plasma cell to secrete the protein. In contrast, the B-cell produces a mature mRNA transcript that encodes a stretch of hydrophobic amino acids that facilitates the protein to be anchored in the plasma membrane. Analysis of some proteins like LDL (low density lipoprotein) –receptor indicates that the various domains of this protein come from different exons probably by a process called exon shuffling. These domains in LDL protein are strikingly similar to other proteins like epidermal growth factor receptor, blood clotting factors and C9 complement factor. 7 Fig. 4C: Processing of different introns Many of the examples cited above suggest that introns of pre-mRNAs contain non-coding regions and are eliminated during splicing. The joining of different combinations of exons in a pre-mRNA transcript produces proteins of different kinds. However this is not true always. DNA sequences that become part of intron sequences of some pre-mRNAs are also shown to code for proteins suggesting that genes are embedded in genes. Examples include the pupal cuticle gene of Drosophila is an intron of another gene that codes an enzyme important in the synthesis of purines (adenine and guanine). While the sequence is excluded in the mature RNA transcript of the purine synthesizing enzyme, it however produces pupal cuticle mRNA of 0.9 kilobase when it is transcribed separately from the DNA sequence. Introns are longer than exons generally, and may a play a role in the transport of processed mRNAs from nucleus to cytoplasm. Mutations in an intron can disrupt the correct splicing and can enhance the transforming activity of certain oncogenes (ex: ras). Introns mark the functional protein regions while exons encode well-defined structural domains in proteins. Addition of 5’ Cap Most of the eukaryotic mRNAs contain a cap structure consisting of 7-methyl guanine, an additional nucleotide at the 5’end. The guanine nucleotide is added soon after the initiation of 8 transcription (even before the completion of the transcription of the mRNA) and is a cotranscriptional event as illustrated (Fig. 5). The guanine nucleotide joins the 5’-end of the mRNA by a 5’-5’ bond instead of a 5’-3’ phosphodiester bond that joins all the other nucleotides. Afterwards, a methyl transferase enzyme adds a methyl group at position 7 of the newly added guanine nucleotide. Also the 2’ OH group of sugars joined to the second and third nucleotides of mRNAs is methylated. The 5’ cap may offer stability to eukaryotic mRNAs and regulates translation by providing a binding site for several factors. Addition of Poly A–tail In addition to the removal of introns, the pre-mRNAs in eukaryotes are processed, at a position from 11-30 nucleotides downstream of an AAUAAA consensus sequence, in their 3’untranslated regions. The cleavage is aided by polyadenylation specificity factor and cleavage stimulation factor. These factors bind to the RNA polymerase during transcription when the polymerase reaches the end of the transcribing gene. After the cleavage, a number of adenine nucleotides are added by the enzyme polyA polymerase in the presence of ATP to the 3’ end of a cleaved mRNA (Fig. 6). The presence of poly A tail may confer stability to mRNA and also helps in the circularisation of mRNA during translation due to an interaction between proteins bound to the 5’and 3’ends of mRNA. The pseudo-circularisation process may facilitate an efficient reinitiation of protein synthesis in eukaryotes (see latter). Fig. 5: Steps in the addition of 5’ Cap in eukaryotic mRNAs RNA editing This is yet another way of modifying the transcribed RNA and is different from splicing. Here nucleotides are added, deleted, or both events can happen. It can occur by chemical modifications as in the conversion of cytosine to uracil by a process called deamination or by a guide RNA. In the editing process, the RNA transcript is modified but not the DNA. For example, apolipoprotein mRNA in some cells produces a full protein with a higher molecular mass. However, the same mRNA has a stop codon in some cells and produces a truncated protein 9 with a lower molecular mass, approximately half of full form of the protein. In the latter, one of the mRNA codons, CAA, that codes for the amino acid glutamine is modified to UAA, a stop codon and produces a truncated form of apolipoprotein. The C to A conversion occurs by cytidine deaminase enzyme. This modification occurs only in certain cell types. For example the editing of apolipo mRNA occurs in intestinal cells and does not happen in liver cells. In some mRNAs, for example, the mRNA that encodes Cox II protein in trypanosomes, four Us are inserted in a specific region that alters the reading frame of the message and the type of protein produced by the mRNA. The editing in this case occurs by a special RNA called guide RNA that consists of 40-80 nucleotides and with the help of other proteins like an endonuclease and a ligase. Fig. 6: Addition of Poly A tail to a eukaryotic mRNA Ribosomes Subunits, Composition and Morphology Free ribosomes are found in the cytoplasm and are involved in the synthesis of cytosolic proteins, whereas the membrane bound ribosomes are associated with the endoplasmic reticulum and are involved in the synthesis of secretory proteins that are destined to reach various organelles. In addition, ribosomes are found in the organelles like mitochondria and choloroplasts. Organelle ribosomes resemble prokaryotic ribosomes. The subunits differ in their size, length, width, composition, and in molecular mass. Protein synthesis occurs on the surface of ribsomes. Ribosomes are RNA-protein complexes consisting of two subunits: a small (30S in prokaryotes and 40S in eukaryotes) and large subunit (50S in prokaryotes or 60S n eukaryotes). Together these subunits form monosomes (70S or 80S in prokaryotes and eukaryotes respectively). When subjected to centrifugal force, the sedimentation value or Svedberg coefficient (S) of the monosomes and their subunits are different. S Values are dependent not only on mass but also the by shape and density. Although S values increase with molecular mass of the ribosomal subunits, the S values of a monosome are not exactly add to its constituent subunits. The S value of prokaryotic monosome is 70S and its constituent subunits are 30S and 50S. The small prokaryotic 30S subunit is divided into head, body and side bulge or platform (Fig. 8). A distinct groove separates the head from the body. This subunit is composed of 10 approximately 21 proteins (S1-S21, S refers to small subunit and they are named serially based on their migration in gel electrophoresis), and 16S ribosomal RNA that play a critical role in the recognition of start codon in prokaryotic mRNAs. The large 50S subunit is 160 kDa approximately and it consists of 34 proteins (L1-L34) and two ribosomal RNAs: 23S (2900 bases) and 5S (120 bases) (Fig. 7). The large subunit is more isometric than the small subunit with a linear size being equal to 200 to 230A0 in all directions. At the periphery, it has three protuberances; the lateral is called the L7/L12 stalk, a central protuberance and a side lobe or L1 ridge (Fig. 8). Electron microscopic observations indicate that the appearance of eukaryotic and prokaryotic ribosomal subunits is identical except that the 40S subunit has a protuberance located on the head and appears to be bifurcated at the end of the body distal to the head. Ribosome’s are not only involved in the actual peptide bond formation but a) provide the necessary platform for the correct positioning of the tuna molecules carrying amino acids or decollated tunas, b) facilitate the movement of tRNA on the ribosome and c) guide the accuracy of the movement. Processing of rRNA Further, the size of the precursor of rRNA molecule is very long and is processed. The ribosomal DNA of E. coli is transcribed as a 30S RNA precursor that is processed to yield 23S, 16S and 5S RNA species. The 23S and 5S transcripts go into the composition of the 50S subunit, whereas the 16S transcript is with the 30S small subunit (Fig. 7). The size of the precursor RNA transcript in mammalian cells is 45S and is cleaved to give rise 28S, 18S and 5.8S RNA molecules. The 28S and 5.8S rRNA eventually become a part of the 60S subunit, and the 18S rRNA transcript becomes part of the 40S subunit. The small ribosomal subunit of eukaryotes also contains 5S rRNA but it is transcribed as a separate entity from a ribosomal RNA gene. The 5S rRNA is present both in pro- and eukaryotic ribosomes and is conserved through evolution. Figs. 7 and 8: Composition and morphology of eukaryotic and prokaryotic ribosomes 11 Functions of ribosomal subunits The small subunit plays a role in the initiation of protein synthesis process, and regulates the fidelity of interaction between the three bases in the mRNA codon- tRNA anticodon. In contrast, the large subunit is the center for the formation of peptide bonds between adjacent amino acids and provides the pathway for the release of nascent or emerging proteins. Ribosomes consist of three sites called aminoacyl (A), peptidyl (P) and the exit (E)- sites that interact with the amino acylated tRNAs, peptidyl tRNA (tRNA carrying growing polypeptide), or with deacylated tRNAs respectively. The distance between these sites is 20A0-50A0. Further, crystallographic analyses of prokaryotic 30S ribosomal subunit reveal that 16S rRNA of 30S subunit modulates the movement of mRNA-tRNA, and the ribosome dynamically changes its shape during different functional states. These facts may explain why the structural features in tRNAs (see latter) are conserved during evolution. Channels in the small subunit of ribosomes facilitate the entry and exit of mRNAs and a channel in the large subunit facilitates the exit of nascent or newly made polypeptide. Thus ribosome is not a static component. It is a dynamic molecular machine with moving parts and a very complicated mechanism of action. Ribosomal RNA, present at the interface between ribosomal subunits, not only plays a role in the structure but also plays a role in two of the most important functions: a) decoding of mRNA and b) addition of peptide bond between adjacent amino acids. Various studies suggest that the 16S rRNA of the small ribosomal subunit scrutinizes the correctness of the codon-anticodon interaction and is involved in decoding the mRNA sequence. The interaction between the first two base pairs of codon-anticodon is checked more rigorously by the nucleotides in 16S rRNA than the interaction between the third base pair. This fact relates to the wobble hypothesis (see genetic code). 16S rRNA plays an important role in identifying the ‘start codon’ in mRNA in prokaryotes (see latter). In contrast, the peptidyl transferase center in the 50S subunit that catalyzes peptide bond formation between amino acids contains 23S rRNA. To perform these functions (decoding and peptide bond formation), the crystal structure data of ribosomes suggest that rRNA is located in the center or at the interface between large and small subunits of the ribosome where as proteins are located mostly peripherally. Peptide bond formation does not consume any additional energy or require ATP hydrolysis. However it uses the energy stored in the acyl bond that was created during the charging of tRNA (see latter). Some of the ribosomal proteins also shield the negatively charged RNA molecules. Polysomes A given messenger RNA may be translated by one or more than one ribosome. Accordingly the number of copies of proteins produced will vary. A messenger RNA that is translated efficiently by several ribosomes can give rise to a polyribosome complex. The total ribosomes in a cell are distributed as small and large subunits (30s and 50S or 40S and 60S), monosomes (70S or 80S i.e., single ribosome bound to mRNA), and polysomes (an mRNA bound by two to several ribosomes). Isolation and analyses of the relative proportion of small subunits, large subunits, monosomes and polyribosomes of total ribosomes reveal to some extent the protein synthetic activity of the cell. Ribosomes isolated from a cell carrying active protein synthesis will have very few subunits and monosomes but will have more polysomes. This is due to an increased rate in the initiation of protein synthesis. Sometimes cells that are defective in protein synthesis particularly at the level of elongation may also show up an increased proportion of polysomes relative to their monosomes. Here the ribosomes pile up on mRNA and are released at a slow 12 rate. Under such conditions, the polysome analyses can be correlated with protein synthesis in vivo. One can estimate protein synthesis in cultured cells (control and treated cells) by monitoring the incorporation of labeled amino acid like [S-35]- methionine or [C14] –leucine into the acid precipitable protein based on the total uptake of the radioactive amino acid. In contrast, a defect in the initiation step of protein synthesis in cells leads to the formation of a high proportion of monosomes relative to polysomes. Transfer RNA (tRNA) Transfer RNAs are small RNAs compared to mRNAs and ribosomal RNAs. The tRNAs act like adaptor molecules recognizing an amino acid on one side and the corresponding sequence information in the mRNA sequence on the other side. Cells thus contain a minimum of twenty tRNAs: one specifying each amino acid. However analyses of tRNAs and the genetic code (see latter) have shown that cells generally contain more than twenty tRNAs and sometimes more than one tRNA specifying an amino acid. Transfer RNAs contain 74-95 nucleotides. However the precursor or the primary transcripts of tRNAs consist of around 125 nucleotides that are processed to yield mature tRNAs. In both prokaryotes and eukryotes, the primary transcripts are longer and contains introns that are processed out to give rise a functional tRNA. The various regions of a typical mature tRNA are shown in Fig. 9. Fig. 9: Transfer RNA with 5′ Phosphate and 3′ -OH group Processing It includes cleavage of the primary transcripts, splicing (joining of exons and removal of introns) and addition of certain bases and modifications of certain bases. The processing is accomplished by enzymes like RNAses P, D and E. As and when the primary transcript is made, it folds itself into the stem-loop like structure. This folding is required and plays a critical role for the nucleases to act upon the precursor molecule to remove certain nucleotides in the 5’ and 3’ends of tRNAs. The processing occurs in an orderly manner and is somewhat different in prokaryotes and eukaryotes. The pre-tRNA molecule in eukaryotes contains a 5’ leader sequence of about 15-16 nucleotides, an intron of 14-15 nucleotides and two additional nucleotides at the 3’end. Removal of these nucleotides yields mature tRNA. The two most important features between the 13 primary and processed transcripts between prokaryotic and eukaryotic tRNAs are a) the absence of intron sequence near the anticodon, and b) the presence of CCA at the 3’end of pre-tRNA in prokaryotes. In eukaryotes, the CCA nucleotides to which the cognate amino acid is attached are added at the 3’end of mature tRNA by the action of nucleotidyl transferase enzyme. Modified bases Both pro- and eukaryotic tRNAs contain unusually rare and modified bases such as ribothymidine, dihydrouridine, pseudouridine, 4-thiouridine, I-inosine, 1-methyl guanosine and N6- isopentenyl adenosine (Fig. 10). The modified bases arise due to the activity of special tRNA-modifying enzymes. Thymidine is generally present in DNA but it is also found in tRNA. Uracil is modified to ribothymidine or pesudouridine by the addition of a methyl group or an amino group respectively. Modifications of tRNA are required to affect the speed and accuracy of protein synthesis. The modifications also play a critical role in maintaining proper reading frame and for the movement of tRNAs from A-site to P-site (see latter). For example lysyl tRNA (tRNALys with an anticodon UUU) recognizes AAA codon in mRNA and undergoes a modification of N6-threonylcarbamoyladenosine at position 37 adjacent and 3’ to the anticodon to bind AAA in the A-site of ribosomal 30S subunit. Figs 10: Modified nucleotides in tRNAs Fig. 11: 3-D structure of tRNA The structures, but not the exact composition of the nucleotide sequences of tRNAs, are similar. The secondary structure or the two-dimensional structure of tRNAs resembles to clover leaf (Fig. 9). Many of the nucleotides are complimentary to each other and form intermolecular hydrogen bonds. As a result the tRNAs assume a typical structure which is critical for their function. The tertiary or 3-D structure of tRNA resembles to L-shaped structure and is a result of nine hydrogen bonds involving base pairing between several invariant residues (Fig. 11). The interactions among bases in the T- and D- arms of tRNA facilitate the folding of the molecule into an L-shaped structure with the anticodon at one end, and the amino acid acceptor at the other end. 14 Charging of tRNAs or Aminoacylation The joining of an appropriate amino acid to the CCA containing 3’ end of amino acid acceptor stem of the tRNA represents aminoacylation or charging of tRNAs. The reaction is catalyzed by an aminoacyl synthetase and requires 2 high-energy bonds of ATP. The amino acylation of tRNA is very specific and occurs in two steps. In the first step, the -COOH or carboxyl group of an amino acid reacts with ATP producing aminoacyl AMP (adenylated amino acid, see Fig. 12) and inorganic pyrophosphate (PPi). In step 2, the activated adenylated amino acid is transferred to the 2’ or 3’ OH group of a sugar linked to an adenine base present at the 3’ end of an appropriate tRNA. In the final reaction, the carboxyl group of the amino acid is linked to the 2’ or 3’ –OH sugar to a base present at the 3’ end of a tRNA. This base is always an adenine (Fig.12). Fig. 12: Aminoacylation of tRNA Each aminoacyl synthetase recognizes specific amino acid and a tRNA. Cells have twenty different synthetases, one for each of the 20 amino acids. The number of tRNAs present in a cell may vary from 30-50 and thus exceed the number of amino acids. This suggests that there may be more than one tRNA for some of the amino acids. These are called isoaccepting tRNAs with different anticodons that accept the same amino acid (see below on wobble). The enzyme aminoacyl synthetase recognizes specific sequences in tRNA particularly present in the D-loop, anticodon loop and in the acceptor stem. For example, if the G3: U70 nucleotides of tRNAala are used to replace the 3:70 base pair of tRNAcys or tRNAphe, then these modified tRNAs are recognized by alanyl tRNA synthestase and charged with alanine suggesting that G3:U70 base pair is a critical identity element in tRNAala for its specific synthetase. Genetic Code It refers to the relation between the four-letter nucleotide sequence information in mRNA to the amino acid sequence information in proteins. The cracking of the genetic code is a history now. 15 Several theoretical considerations and elegant experimental results supported that a group of three nucleotides, called a codon, specifies an amino acid. An mRNA that contains all of its information in 4 letter nucleotides will have then 64 codons (43 = 64) that specify the twenty naturally occurring amino acids. In contrast, a single nucleotide or groups of two or four nucleotides would provide a maximum of 4, 16 or 256 codons respectively. While the single and double letter codons cannot represent all of the twenty amino acids, the codon containing four nucleotides was also not supported experimentally and theoretically as it uses maximally of the four letters to specify twenty amino acids compared to the triplet code. In fact, the very early mutagenesis experiments provided evidence to support the triplet code. It was shown that bacteriophage T4 was unable to tolerate genetic changes that probably have altered one or two bases. However, the phage was able to tolerate an alteration (deletion or insertion) of three bases. Based on such genetic experiments, it is suggested that the genetic code is a triplet code. In a triplet code, a change in one or two bases (insertion or deletion) alters the reading frame of the mRNA and all the subsequent amino acids that are incorporated into the protein to the right side of deletion (-) or insertion (+) (that is on the –COOH side of protein). However insertion of three nucleotides into an mRNA sequence would change the coding ability of the message only of those triplets at, and between the insertion of bases but not the amino acid sequence downstream of the three inserted bases (see latter frame-shift mutations). Cell-free translational systems The genetic code was in fact deciphered before the cellular messenger RNAs were purified. However by then cell-free translational systems were shown to support protein synthesis of the endogenous mRNAs in vitro. The proteins prepared by such cell- free extracts can be labeled by supplementing one radioactively labelled amino acid along with the other nineteen unlabelled amino acids. The protein synthesis of the extracts carried out by endogenous mRNAs, or by exogenously supplied mRNAs can be monitored by the incorporation of radioactively labeled amino acid into the acid precipitable protein. This is possible because the cell-free protein synthesizing systems contain all the machinery viz., the ribosomes, transfer RNAs, aminoacyl synthetases that catalyze the joining of amino acids to tRNAs and other soluble factors (initiation, elongation and termination factors) required for the protein synthesis. Additionally the extracts are supplemented with ATP, GTP, and energy generating enzymes along with salts like K+ and Mg2+. Such cell-free translational systems derived from bacterial extracts eventually provided a means to determine the amino acid sequence of proteins coded by artificial and natural templates. Subsequently these cell-free translational systems have been used as a source for the preparation of reconstituted lysates to identify defective components of translational machinery, for the purification of protein factors and enzymes, to study the mechanisms of antibiotic actions, and to determine the mechanics and regulation of cytosolic and secretory protein synthesis. Since many proteins are processed in physiological conditions and is difficult to know the sequence if any in prepro-proteins (unprocessed proteins), translation of such mRNAs in vitro systems devoid of the peptidases or proteases has provided a means to determine the sequence of full length proteins. In addition, these translational systems are found useful to evaluate the effects of antibiotics, toxins, and a host of other novel compounds for their ability to affect protein synthesis at specific steps. While bacterial systems and rat liver cell-free translational systems are used initially, subsequently the preparation of heme-deficient rabbit reticulocyte lystates and cell-free 16 translational systems derived from wheat embryos have unraveled some of the crucial regulatory mechanisms involved in the initiation and elongation of mammalian cytosolic and secretory protein synthesis. Wheat germ lysates, that do not have any endogenous mRNAs, were used to determine the full length nature of a secretary protein, the role of a signal peptidase that processes the signal sequence of a secretory protein, and also the role of other regulatory proteins like signal recognition particle (SRP) that inhibits secretory protein synthesis, and docking protein that relives the inhibition mediated by SRP. Similarly heme-deficient rabbit reticulocyte lysates that carry mainly translational apparatus and mostly endogenous globin mRNA were used to determine the importance of heme in the regulation of globin protein synthesis and to elucidate the mechanism of inhibition of general translation in reticulocyte lysates in hemedeficient lysates. Micrococcal nuclease-treated reticulocyte lysates can also be used to determine the translational products and activity of exogenously supplemented mRNAs. Micrococcal nuclease is activated in the presence of calcium and can degrade the endogenous mRNA. Before supplementing the exogenous mRNA, these nuclease treated lysates have to be treated with EGTA that chelates calcium and inactivates the enzyme. While many animal systems can yield cell –free translational systems with varying efficiencies, plant cell-free translational systems are difficult to obtain because of the presence of vacuoles that contain hydrolytic enzymes that can damage RNA. Synthetic mRNA templates The preparation of artificial template mRNAs is yet another important milestone in understanding the genetic code. This has become possible because of the discovery of polynucleotide phosphorylase enzyme. This enzyme is found to catalyze in fact the degradation of RNAs to the corresponding ribonucleotides and inorganic phosphate in vivo. But, interestingly, the purified enzyme has been observed to catalyze the formation of ribonucleoside triphosphates (rNTPs) in the presence of high concentration of ribonucleoside diphosphates in vitro. The incorporation of a ribonucleotide (A, U, G or C) is however dependent on the relative amounts of the nucleotides used in the reaction mixture. For example, the chances of the incorporation of nucleotide A (adenosine) only once in the template molecule in a reaction mixture consisting of A and C in the ratio of 1: 4 are 1/5 whereas the chances for the incorporation of C are 4/5, the highest. In other words, a template molecule with AAA will be synthesized least and CCC will be the most abundant. Templates with two C s and one A (CCA, ACC and CAC) will be the next abundant compared to two As and one C (AAC, CAA and ACA). The codon sequence of these templates containing a mixture of nucleotides could determine to some extent based on the relative abundance of the proteins produced by these templates in cell-free translational systems, and then identify the amino acid sequences of the protein (see below). However this method alone cannot give a precise sequence of information of codons in mRNAs until additional information is available from other methods. Amino acid analyses of polypeptides produced by synthetic templates Synthetic mRNAs with single nucleotide or mRNAs (such as AAA…, CCC…, UUU…, and GGG….), with alternating nucleotides (ACACACACACAAC), and with repeating sequences (AACAACAACAACAACAAC) yielded a good amount of information to relate the nucleotide sequence to the amino acid sequence in proteins. For example, translation of synthetic templates containing single nucleotides (homopolymers) such as A, C, U or G in cell-free translational 17 systems yielded polypeptides that contained a single amino acid sequence like lysine in the case of polyA, phenylalanine in the case of poly U and proline in the case of poly C. Translation of poly G is however found to be weak and produces polyglycine. The reason appears to be that the poly G mRNAs lack proper folding. Translation of templates containing alternating nucleotides like ACACACACAAC, yielded a polypeptide sequence that contains threonine and histidine amino acids alternatively. To determine whether the codon sequence ACA or CAC is the cause for the incorporation of threonine or histidine into the sequence, templates with repeating trinucleotides such as AACAACAACAACAAC have been made and translated in cell-free translational systems. Such sequences produced three types of polypeptides containing only asparagine (Asn), threonine or glutamine. This is possible if the start site in the mRNA in each of these cases is different. Now we know most of the natural mRNAs unlike synthetic mRNAs have a ‘start’ site and ‘stop’ site. It is likely that if the synthetic mRNA sequence in the above case is read as AAC AAC AAC AAC AAC, it may give a polypeptide that contains only Asn. But if the first A is skipped, then the message is read as ACA ACA ACA ACA AC, and if the first two As are skipped then the message will have a different sequence like CAA CAA CAA CAA C. The common amino acid that is incorporated when the mRNAs containing AC repeats and AAC repeats is threonine. Based on these results it is suggested that probably the ACA codon is responsible for the incorporation of the amino acid threonine. However these results required further validation. The reading frame in natural mRNAs is set by start codon which is usually AUG and the code is generally nonoverlapping or in other words each nucleotide in an mRNA belongs to a single reading frame. Codon, charged tRNA and ribosome complexes can be captured on a filter Researchers who were in the race to crack the genetic code had yet another interesting observation wherein they found that a small triplet codon (like AUG, AAA or CUG etc.,) can attract a complementary anticodon of a tRNA carrying the amino acid. The codon and anticodon together can bind to ribosomes. Such codon-charged tRNA-ribosome complexes can be captured on a filter paper. The complex can be identified because the amino acid bound to tRNA in the complex is radioactive. This technique is found to be relatively more informative in deciphering the genetic code because it is easy to prepare and handle small triplet nucleotide sequences than long RNA templates. The synthetic triplet codons were incubated with a mixture of tRNAs each carrying a radioactive amino acid. Small molecules like the tRNAs carrying amino acids whose anticodons are not complementary to the codons cannot bind to the codon in the reaction and will pass through the filter. This technique really facilitated to identify the recognition of 61 triplet codons that are now known to recognize the twenty naturally occurring amino acids. Wobble and degeneracy Out of the total 64 codons, 61 codons specify 20 amino acids, and three codons UAA, UAG and UGA specify stop codons. Since 61 codons specify the 20 naturally occurring amino acids, it suggests that there are many amino acids that are specified by more than one codon. Hence it is suggested that the genetic code is degenerate. Based on the number of sense codons, it is suggested that there should be 61 tRNAs recognizing the 61 sense codons. However purification and characterization of tRNAs revealed that many purified tRNAs are also found to recognize a) more than one amino acid, and b) tRNAs contain some modified bases like inosine (derived from adenine) in the anticodon that is different from the regular four bases present in RNAs. Transfer RNAs with different anticodons that accept the same acid are called isosccepting tRNAs. Based 18 on these observations, it is suggested that the first base in the 5’-anticodon of tRNA does not follow strict Watson-Crick base-pair rule position while pairing with the 3’ end of codon. This imperfect or nonstandard base pairing called wobble occurs between the third position of the codon in mRNA and the first position of the anticodon in the tRNA (Fig. 13). Indeed analyses of all the codons through the filter binding assays did show that 18 of the twenty amino acids have more than one codon. Two of the amino acids, viz., methionine and tryptophan are specified by one codon each. Among the others, three amino acids (serine, leucine and arginine) have 6 codons each accounting 18 of the 61 codons. Five of the amino acids (glycine, proline, alanine, valine and threonine) have 4 codons each, and nine other amino acids (phenylalanine, tyrosine, cysteine, histidine, glutamine, glutamic acid, asparagines, asparatic acid and lysine) are specified by two codons. Isoleucine has three codons, and three codons UAA, UAG and UGA specify stop codons thus accounting the 64 codons ( 2 + 18 + 20 + 18 + 3 + 3 = 64) in the genetic code (Fig. 14). Fig. 13: Wobble Indeed, analyses of many of the codons that specify the same amino acid have shown that these codons differ in their third position. For example the codons specifying glycine are GGG, GGC, GGA and GGU. In these cases, it is the last nucleotide residue or the nucleotide residue in the third position of the codon is different. The degeneracy of the code or wobble may be economical because it reduces the number of tRNAs from 61 to a minimum of 30 that are required to recognize all the 61 triplet codons. The estimated number of tRNA species to be present in bacteria is 30-40 and the number may be somewhat higher in some eukaryotes. The code is mostly universal with the exception of few of the mitochondrial and protozoan codons that behave slightly differently. For example mammalian mitochondrial UGA codes for 19 trp instead of a stop signal. AUG codon is used in the cytoplasm to specify methionine during initiation and elongation steps of protein synthesis. However AUA that specifies isoleucine in the cytoplasm is used to specify internal methionine or methionine during the elongation step in mitochondria. AGA and AGG codons that specify Arg amino acid in the cytoplasmic code however specifies a stop signal in mitochondria. In fruit fly, AGA and AGG specify a serine codon but not a stop codon or arginine. Again an analysis of the changes in coding specificity of different codons reveals that a change in the base occurs primarily at the third position of the codon in mRNA. Such changes may facilitate to decrease the number of tRNAs. Fig. 14: Genetic Code Mutations Changes in the nucleotide sequences alter the genetic code. Point mutations arise because of changes in a single nucleotide which are different from other kinds of drastic changes that occur in DNA due to extensive insertions and deletions. Missense mutation / Substitution mutation A change in one codon (trinucleotide sequence) that specifies a particular amino acid to another codon that specifies a different amino acid can occur by a single base change. The best example is sickle cell anemia, a human genetic disease. Here the glutamic acid at position six in the human β-globin is replaced by valine. The change is the result of a base change at the second position of the codon GAA that codes for glutamic acid to GUA that codes for valine. 20 Nonsense mutation A change in the base of a codon specifying an amino acid to another base that results in a stop codon. If it happens in the middle of a genetic message, it results in premature termination and incomplete polypeptide synthesis. Silent mutation Often a change in the base that is present in the third position of a codon of an mRNA to another base can result in a synonym that does not alter the amino acid sequence of the encoded protein. Such changes are called silent mutations. Neutral Mutation It is a kind of missense mutation where the change in a base leads to a change in the incorporation of a different amino acid that is chemically similar to the original one, and does not influence the protein function. Frame shift mutations Insertion or deletion of one or more base pairs in the gene sequence affects the reading frame of mRNA. As a consequence the amino acid sequence of the protein from the point of mutation to the C-terminus will be modified. For example 5’…. ABC ABC ABC ABC ABC ABC ……3’ sequence in mRNA is modified by the insertion of a D for example can give a rise a message to ABC ABC DAB CAB CAB CAB C. Even deletion of a base also alters the coding capacity not only at the site of modification but also the rest of the message from the site of modification. However in the above sequence if insertion or deletion of three bases (like D, E and F) instead of one or two bases occurs then the coding specificity of the message is altered at and between the insertions of these bases but the downstream sequences are not altered as shown below. 5’…. ABC ABC ABC ABC ABC ABC ……3’ is changed to 5’…. ABC DAB CAE BCA FBC ABC ABC ……3’. Suppressor Mutations A suppressor mutation as the name indicates, suppresses the influence of another mutation. Thus the organism that contains a suppressor mutation is a double mutant and it has two mutations: one the original mutation and the second one being the suppressor mutation that will produce a phenotype which is similar to the wild type. However this is not called reverse mutation. In reverse mutation, whatever change occurred in the original nucleotide is restored. A suppressor mutation occurs away from the original mutation site. The suppressor mutation can occur within the same gene that contains already a mutation (intragenic suppressor) or in another gene (intergenic suppressor). Both kinds of suppressor mutations block the effects of an earlier mutation. Gene density and overlapping genes While the genome size and the approximate number of total genes present in an organism is related to the complexity of an organism, gene density that refers to the average number of genes per million bases (Mb) of genomic DNA is however high in lower organisms. An analysis of gene density in different organisms reveals that highest gene densities are found in viruses where 21 sometimes both strands of DNA are used and encode overlapping genes. In bacteria, the gene density is very high compared to eukaryotes. Overlapping genes are found in many viruses where the genome size is small. Such overlapping genes produce more than one protein depending on the reading frame of the gene and the start site. In prokaryotes, the mRNAs are polycistronic, and in such cases the ribosomes have to be able to find the start codon before they are detached. In eukaryotes, alternative RNA splicing is used to generate variant proteins from one gene. Protein Synthesis The machinery of protein synthesis consists of ribosomes, subunits, mRNAs, tRNAs and amino acyl synthetases, protein factors, amino acids, ATP and GTP. For convenience, the process is divided into four steps: initiation, elongation, termination and ribosome recycling. Although, the overall process of protein synthesis is similar, several differences exist among prokaryotes, archea and in eukaryotes. The description here mostly refers to the general protein synthesis and to some important differences between prokaryotes and eukaryotes. Initiation Initiator and elongator methionyl tRNAs and formylation Initiation of protein synthesis in vivo is a very precise process. In spite of several nucleotides present in the 5’end of mRNA, ribosome skips these codons and initiates the synthesis from the first AUG codon that it encounters on the 5’ side. Since AUG codes for methionine, it is the first amino acid to be incorporated in any protein. Rarely GUG may be used that codes for valine. However two tRNAs are involved in bringing the amino acid methionine: one is initiator methionyl tRNA (fMet-tRNAfMet in prokaryotes) and another one is elongator methionyl tRNA(Met-tRNAmMet).The difference is that the methionine bound to the initiator tRNA in prokaryotes is formylated by a transformylase enzyme and the formyl group comes from N10 tetrahydrofolate. The methionine carried by the elongator tRNA is not formylated. Athough two tRNAs exist in eukaryotes for methionine as in prokaryotes, the methionine bound to initiator is not formylated in eukaryotes. The sequence differences between the three tRNAs are illustrated in Fig. 15. Further, IF2 initiation factor in prokaryotes and eIF2 in eukaryotes facilitate the joining of initiator tRNA carrying methionine to the respective small ribosomal subunits in the presence of GTP. The elongator methionyl tRNA (Met-tRNAmMet) and other amino acylated tRNAs are delivered to the 70S initiation complexes by elongation factor EF.Tu in prokaryotes and by EF1 in eukaryotes. Formation of 70S initiation complex The process of initiation occurs in an orderly manner with the help of initiation factors (IFs) (Fig. 16). While the prokaryotic initiation factors are called IFs the eukaryotic factors are represented as eIFs. The final product of initiation step is the formation of a 70S in prokaryotes and 80S ribosome complex in eukaryotes with the corresponding initiator tRNA properly positioned on the first AUG codon of mRNA at the P site of ribosome. Initiation in prokaryotes starts with the small ribosomal subunit (30S) that is bound by IF1, IF2 and IF3 factors. The IFs interact with domains in small ribosomal subunit that will subsequently interact with the amino acylated, peptidyl and deacylated tRNAs respectively i.e., A site, P site or E site in ribosome. While IF1 binds to the A site in the ribosomal subunit that will eventually harbor an incoming amino 22 acylated tRNA, IF2 interacts with the A and P sites, and IF3 interacts with E site of the ribosome. To begin with, the f-Met-tRNAfMet joins IF2.GTP associated with the 30S subunit in a codon-independent manner. The 30S complex also binds mRNA through its ribosome binding site (RBS). Although in eukaryotes, the Met-tRNAiMet joins the 40S ribosome before the mRNA joins, the order of these two events in prokaryotes is not clear. RBS is also called as ShineDalgarno sequence in prokaryotes. It is a 7-8 nucleotides long sequence in mRNA located upstream of the initiation codon (AUG). It contains a polypurine rich sequence (Ex: 5………..AGGGGAAAU--- AUG……..3’ in E. coli trpA gene ) that interacts with the polypyrimidine sequence (3’ ….CCUCCU…..5’) present in the 16S ribosomal RNA of the 30S subunit through complementary base pairing. This will facilitate 30S subunit of ribosome to attach the mRNA and to position itself over the initiation codon directly. Fig. 15: Important sequence differences in different Methionyl tRNAs However this unstable 30S complex (30S ribosome.IF1.IF3.IF2.GTP.fMet-tRNAfMet. mRNA) undergoes a stable conformation that promotes codon-anticodon interaction. This triggers a conformational change in 30S ribosome and facilitates the release of IF3 (an anti-association factor) so that the 50S subunit joins the 30S initiation complex to form 70S initiation complex. A GTPase facilitates the hydrolysis of GTP bound to IF2 to GDP and that will facilitate the release of both IF2 and IF1 from the 30S subunit. At the end of initiation, 70S ribosome complex devoid of initiation factors is formed with fMet-tRNAfMet positioned in the P-site of ribosome at the start codon (Fig. 16). Functions of Initiation Factors All three initiation factors help to position the initiator tRNA in the ‘P’ site of ribosome on the start AUG codon in mRNA. IF3 especially stabilizes the 30S initiation complex. IF1, the smallest protein factor among all the three initiation factors (8.2 kDa in E. Coli) promotes the interaction between IF2 and 30S subunit and more specifically the interaction of IF2. fMet. 23 tRNA.GTP with the initiation codon of the mRNA in the P-site. IF1 through its interaction with the A-site blocks the association of amino acylated tRNAs to the A-site until the formation of 70S initiation complex. IF2 is the largest initiation factor (approx 89-90 kDa) with highest affinity to ribosomes compared to the two other factors and joins specifically fMet-tRNAfMet. The complex IF2.GTP will promote the association of 30S initiation complex with 50S subunit to form 70S initiation complex. Hydrolysis of GTP bound to IF2 by a GTPase occurs in the presence of ribosomes. It is not clear whether the GTPase activity is intrinsic to IF2 protein and is triggered upon the association of 30S pre-initiation complex with the 50S subunit. The GTP hydrolysis however at the end of initiation facilitates a) the release of IF2.GDP from the 30S initiation complex, and b) the adjustment of initiator tRNA in the P-site of ribosome with proper codon-anticodon pairing. GTP hydrolysis by a GTPase however occurs only after the subunits are joined. Hydrolysis of the GTP may cause a conformational change in ribosome that would facilitate the release of all the factors. IF2.GDP that is produced at this stage has reduced affinity for ribosome and is also released along with IF1. IF2 like factor is also recently discovered in eukaryotes and is called eIF5B with a GTPase activity. It plays a role in the joining of large subunit with the small subunit at the end of initiation in eukaryotes (see latter). IF3 is a 20.4 kDa protein. IF3 bound 30S subunit cannot join the 50S subunit to form 70S initiation complex unless the factor is released. Thus, it prevents association of the ribosome subunits. It also promotes the dissociation of noninitiator aminoacylated tRNAs binding to the P-site of 30S ribosomal subunit, the dissociation of 70S complexes, and in the recycling of ribosomal subunits at the end of synthesis. Fig. 16: Initiation of Protein Synthesis in Prokaryotes Elongation This occurs in three steps: 24 (i) (ii) (iii) delivery of the amino acylated tRNA to the A site of 70S ribosome complex; peptide bond formation between adjacent amino acids by petidyl transferase (ribosomal RNA or ribozyme); and the movement of mRNA by three nucleotides. i. EF.Tu/Ts factor promotes the joining of amino acylated tRNA to the A-site in ribosome In step1, an aminoacylated tRNA attachment to the A site in 70S ribosome is catalyzed by elongation factor EF.Tu and GTP. Unlike IF2, EF.Tu has a high affinity for GDP. However GDP inhibits the joining of aminoacylated tRNA to EF.Tu. Hence exchange of GTP for the bound GDP is critical. This GDP/GTP exchange is catalyzed by a guanine nucleotide exchange factor called EF.Ts. EF.Tu binds the 3’end of tRNAs and protects the attached amino acid from entering into the peptide bond formation. After the delivery of aminoacylated tRNA into the A site of ribosome, the GTP bound to EF.Tu is hydrolyzed and the EF.Tu.GDP is released. The latter is recycled by EF.Ts and GTP to form EF.Tu.GTP that is competent to join aminoacylated tRNA. The GTPase activity that hydrolyzes the GTP bound to EF.Tu is stimulated when the ternary complex, EF.Tu.GTP. aa tRNA, joins the ‘A’ site of the ribosome and interacts with the factor binding center of the ribosome. Further the efficiency of GTPase activity is high and dependent on the correct base pairing between codon-anticodon. Thus it acts as one of the mechanisms to ensure proper codon-anticodon interactions. ii.Ribosomal RNA of the large subunit promotes the Peptide bond formation The next step in elongation is the formation of peptide bond between adjacent amino acids. It occurs at the peptidyl transferase center present in the large subunit. The peptide bond formation takes place between the amino acid present at the 3’ end of tRNA of the growing polypeptide chain in the ‘P’ site and the aminoacylated tRNA present in the ‘A’ site. In this process, the growing polypeptide attached to the peptidyl tRNA is transferred to the tRNA present in the Asite (contains a newly arrived amino acylated tRNA). The peptide bond formation requires Nterminus of the protein to be synthesized before the C-terminus. After the addition of peptide bond by the peptidyl transferase center of the large ribosome subunit, the peptidyl tRNA is deacylated. For a long time it is believed that one of the proteins of the large subunit is involved in the catalysis of peptide bond formation. However the current evidence suggests the large size ribosomal RNA (23S in prokaryotes and 28S in eukaryotes) of the large ribosomal subunit catalyzes the peptide bond formation. Although the exact mechanism is yet to be determined, it is suggested that the nitrogen of the nucleotides that accepts a hydrogen atom from the α-amino group of the amino acid bound to amino acylated tRNA can act as a strong nucleophile and can attack the carbonyl group of the growing polypeptide attached to the peptidyl tRNA (as shown below). Since no fresh energy input is required to promote peptide bond formation, the reaction is driven by high energy acyl bond that is formed during the tRNA charging (Fig.17). iii. Translocation of tRNA and mRNA are promoted by EF-G After the formation of peptide bond, the tRNA in the ‘P’site is deacylated and the growing polypeptide chain joins the ‘A’ site. Then the mRNA moves by three nucleotides to bring in the next codon. The deacylated tRNA moves to the ‘E’ site whereas the growing polypeptide chain located at this point in ‘A’ site moves to the ‘P’ site thus allowing room for a new amino acylated tRNA to enter the ‘A’ site. The evacuation of deacylated tRNA from the E-site and the 25 movement of mRNA by one codon relative to the ribosome requires the mediation of elongation factor-G (EF-G) and GTP. EF-G thus acts like a translocase enzyme and drives the translocation of tRNA and mRNA. The translocation step requires the hydrolysis GTP bound to EF-G. The hydrolysis of GTP is stimulated when EF-G contacts the factor-binding center of the ribosome. GTP hydrolysis triggers a conformational change in the ribosome that facilitates the translocation of the growing polypeptide chain from A site to P-site and also formation of EF-G.GDP. In fact, the latter has reduced affinity to ribosome compared to EF-G.GTP complex. EF.G.GDP thus formed replaces the tRNA in the A-site to P-site and occupies the A-site. This will also facilitate the movement of deacylated tRNA from P-site to E-site. This movement of tRNA in the A-site mediates the movement of mRNA by one codon or three bases This movement of mRNA during translocation has been further supported by the fact that movement of rare frame shift tRNAs that have four nucleotides in their anticodon region can move the mRNA by four base pairs. Thus the translocation step is promoted by EF-G.GDP complex that binds to the A-site. Interestingly, EF.G.GDP structure appears to resemble EF.Tu.GTP.aa.tRNA complex which also binds to the A-site. This is a kind of molecular mimicry (Fig. 18). Fig. 17: Peptide Bond Formation iv. Energy requirements in the elongation cycle The elongation cycle consisting of joining of charged aminoacylated tRNA to the A-site, peptide bond formation and translocation of tRNA and mRNA requires the consumption four high energy bonds in each round of elongation cycle or for the incorporation of one amino acid. Two of these high energy bonds come from one molecule of ATP and are used in the charging of amino acid to tRNA, and two of them come from two molecules of GTP. The high-energy bonds of GTP are used in the joining of amino acylated tRNA to the A-site in ribosome, in the fidelity of translation and in the translocation step. In contrast, a minimum of three high-energy bonds is required in the initiation cycle of prokaryotes. Two of these bonds that come from ATP are used in the charging of initiator tRNA and one molecule of GTP is hydrolyzed soon after proper codon-anticodon interaction, before the release of the factors and the formation of 70S initiation complex. In eukaryotes, however there appears to be additional energy requirement to unwind the secondary structure in mRNA and in the subunit- joining step as mentioned above. 26 Termination Release factors (RFs) Two classes of release factors (RFs) have been identified. Class1RFs identify stop codons and facilitate the release of newly made polypeptide chains. When a ribosome reaches one of the three stop codons in mRNA, no amino acylated tRNA enters its A-site. Instead termination factors designated as release factors (RFs) recognize the three stop codons (Fig. 18). Prokaryotes contain two class 1 factors: RF1 and RF2 that recognize three stop codons. RF1 recognizes UAG stop codon, whereas UAA stop codon is recognized by both RF1 and RF2. The third stop codon, UGA, is however recognized by RF2. Unlike in prokaryotes, eukaryotes contain only one factor called eRF1 that recognizes all the three stop codons. Fig. 18 Class1 release factors The class1 release factors carry evidently two functions: one of them is the recognition of the stop codon and the second function involves the release or hydrolysis of the nascent peptidyl chain. A sequence of three amino acids (SPF i.e serine-proline and phenylalanine) in the RF protein recognizes the stop codon and thus serves as a peptide anticodon. The other function (release of the nascent polypeptide) evidently requires a conserved GGQ (glycine-glycineglutamine) sequence in RF. These two regions are close to each other in the absence of a ribosome but in the presence of a ribosome, the release factor undergoes a conformational 27 change that would space these two regions appropriately to serve the two functions as mentioned below. Class II RFs Under the category of class II, only one release factor is identified and is designated as RF3 in prokaryotes and eRF3 in eukaryotes. Class II factors stimulate the dissociation of class I factors from the ribosome and they require GTP. Ribosome recycling After the release of polypeptide chain and RFs, the ribosme is bound by mRNA and deacylated tRNAs in the P and E-sites. The dissociation of ribosomes into subunits, the removal of deacylated tRNAs and mRNAs is aided by ribosome releasing factor, RRF as has been demonstrated in prokaryotes. It resembles to tRNA and thus it can join the A-site. Elongation factor, EF.G, that mimics like a tRNA joins the RRF bound to ribosomes. The binding of these factors somehow facilitates the evacuation of deacylated tRNAs, subunit dissociation and mRNA release. Interestingly, initiation factor 3 (IF3) may also join the small subunit and prevents the joining or association of the large subunit with the small subunit and aids in mRNA dissociation (Fig. 18). Differences in the initiation between eukaryotes and prokaryotes The basic steps in the initiation of protein synthesis are the same in prokaryotes and eukaryotes, i.e., to bring the initiator tRNA and mRNA onto the small ribosomal subunit, identification of start codon, and the joining of large subunit with the preinitiation complex to form 70S or 80S initiation complex. The components and the mechanisms however are more complicated in eukaryotes than in bacteria. In eukaryotes, the methionine bound to the initiator tRNA is not formylated and the steps joining the initiator tRNA and mRNA appear to be occurring one after another. In eukaryotes, there are a dozen protein factors involved in the initiation that are listed in table-1 Eukaryotic initiation factor 2 (eIF2) like its prokaryotic counter part IF2, facilitates the joining of methionyl initiator tRNA to 40S subunits that is bound by a multisubunit eIF3 (an anti association factor) and eIF1A and eIF1. The whole complex is now called the multifactor complex. However, eIF2 has a very high affinity for GDP than for GTP in the presence of physiological concentrations of Mg2+and GDP inhibits the joining of eIF2 to initiator tRNA carrying methionine. Hence eukaryotic cells have a GDP/GTP exchange factor called eIF2B that catalyzes the exchange of GTP for GDP bound to eIF2. The multifactor complex then joins mRNA bound by eIF4F complex. This complex contains three different proteins viz., eIF4G, eIF4E and eIF4A with different functions. While the complex containing three proteins is required for the translation of eukaryotic mRNAs that contain 5’cap, eIF4A with the energy coming from ATP hydrolysis acts as a helicase and facilitates the unwinding of any secondary structure in mRNA. The mRNA is held in place by eIF4E and eIF4G is a multivalent adaptor complex interacting with eIF4E, 4A, eIF3 of multifactor complex and also with the polyA binding protein that binds the poly A tail of 28 eukaryotic mRNAs. In prokaryotes, no ATP hydrolysis occurs to unwind the mRNA structure as in prokaryotes. The first ATP hydrolysis occurs in all organisms is at the charging of initiator tRNA. In addition, the eukaryotic mRNAs do not have anti- Shine-Dalgarno sequence that allows easy recognition of the start AUG on mRNA by 18S rRNA. Eukaryotic mRNAs have 5’ caps and 3’ poly A tails because of which the joining of 43S complex (40S ribosome-Met-tRNAiMet complex bound by several factors) is more complicated than in prokaryotes. An analysis of the sequences around the initiation sites in eukaryotic mRNAs (plants, yeast and mammals) indicates an A preceding the start AUG and G after the start AUG (5’ …….A…AUGG……3’ sequence around the initiating AUG in eukaryotic mRNA). In mammals, the recognition of AUG start codon critically requires the surrounding sequence, GCC (A/G) CC AUG G. eIF1 and eIF1A are the factors implicated in identifying the initiation codon selection and in maintaining the translational fidelity. Further, in eukaryotes, the proper positioning of the initiator tRNA anticodon on the start codon of 48S initiation complex stimulates the GTPase activity of associated with one of the subunits of eIF2 by a protein called eIF5. Hence the latter is called GTPase activating protein (GAP) that hydrolyzes GTP bound to eIF2 to GDP and releases eIF2.GDP. However the 60S subunit joining to 48S initiation complex in eukaryotes requires yet another GTP hydrolysis that is promoted by eIF5B, a factor that has sequence homology with prokaryotic IF2. In addition to 5’ m7GpppN (methylated guanosine cap) and 3’ poly A tail that promote translation, many eukaryotic mRNAs carry secondary and tertiary structural elements like hair pins and pseudoknots respectively, internal ribosome entry sequences (IRESs) at the 5’end that facilitate cap-independent translation, small upstream open reading frames (uORFs) that act normally as negative regulators of translation from the main ORF, and also special nucleotide sequences both at the 5’ and 3’ ends of mRNA that can bind to RNA or proteins. These sequence elements in mRNAs regulate translation. In the elongation cycle, eukaryotes have eEF1A and eEF1B (α, β and γ) are comparable to their function to prokaryotic EF-Tu and –Ts respectively whereas eEF-2 in eukaryotes is comparable prokaryotic EF-G. Further the activity of many of the eukaryotic, but not prokaryotic, initiation and elongation factors is regulated by phosphorylation-dephosphorylation. Average rates of amino acid incorporation in a bacterium like E. coli will be 15-20 aa /sec which is equivalent to a decoding of 15-20 codons or 45-60 nucleotides in an mRNA/sec. Apparently the rate is far slower in eukaryotes, and is about 5-10aa /second at370C Inhibitors Antibiotics (produced by bacteria and yeast and their man made versions that are used to treat various bacterial infections), toxins (proteins produced by pathogenic bacteria such as cholera, diptheria etc.,), lectins (carbohydrate-binding proteins originally found in plants) and physiological agents such as interferons (natural proteins produced in response to viral infection in human cells) inhibit protein synthesis at the initiation, elongation or at termination level. The ribosome is an important target for a wide variety of antibiotics. Broad spectrum antibiotics such as streptomycin and tetracycline were of great clinical importance when they were first 29 discovered. However with the increased resistant strains of bacteria, there is a real need to understand details of how these antibiotics interact with the ribosome. Many of the antibiotics primarily bind to ribosomal RNA and inhibit protein synthesis. Most of the clinically useful antibiotics target the large subunit 50S in the peptidyl transferase center, its vicinity or at the entrance to the ribosome tunnel. Some of the antibiotics bind to the 16S rRNA of the 30S subunit and still interfere with the translocation step than initiation step as mentioned below. Table 1: Translational initiation factors in eukaryotes and their functions Eukaryotic initiation factor Function eIF1 eIF1A ‘Start’ AUG codon recognition Multiple roles? Facilitates Met-tRNAiMet binding to 40S subunit Facilitates the joining of Met-tRNAiMet to 40S subunit, has higher affinity for GDP. Contains GTPase activity, interacts with mRNA and the other proteins. GDP/GTP exchange factor. Functionally it is like Ts factor in prokaryotic elongation cycle. Exchanges GTP for GDP bound to eIF2. Recycles in active eIF2.GDP to active eIF2.GTP so that eIF2 joins initiator tRNA. Promotes Met-tRNAiMet and mRNA binding to 40S. Helicase, unwinds mRNA secondary structure Promotes the helicase activity of eIF4A Binds 5’ cap of the mRNA eIF2 (three subunit protei: α, β and γ) eIF2B (heteropentameric ) eIF3 (Multi subunit protein) eIF4A eIF4B eIF4E eIF4F eIF4G eIF5 eIF5B PABP Complex of cap binding proteins: eIF4A, 4B, 4E and 4G Scaffolding or multivalent adaptor protein that interacts with proteins bound to 5’ and 3’ end of mRNAs. Involved in AUG recognition and activates the GTPase activity of eIF2. In the joining of 60S to 48S complex to form 80S initiation complex. Its sequence resembles to IF2 that is involved in Met-tRNAfMet binding. Poly A- binding protein that provides contact for eIF4G so that mRNA can be circularised 30 Streptomycin interacts with the phosphate backbone of 16S rRNA and S12 protein and hypothesized to increase the binding of non cognate-tRNAs that would lead to misincorporation of wrong amino acids into the polypeptide chains. Tetracycline was found to be a multi-site antibiotic with inhibitory action. It interferes with the A-site tRNA binding. Tetracycline binds to two functional sites in the 30S subunit. It has a major binding site at the A site of tRNA, and a minor binding site near 16S rRNA. It is suggested that the binding of tetracycline interferes in the binding of amino acylated tRNA at the A-site during translation. In the presence of tetracycline, GTP hydrolysis of the ternary complex, EF.Tu.GTP.aatRNA occurs in an unproductive manner and the amino acylated tRNA is ejected from the A-site without peptide bond formation due to a stearic clash with the antibiotic. Tetracycline thus prevents protein synthesis but facilitates GTP hydrolysis. The GTP hydrolysis without protein synthesis is energetically very expensive to the cell. Consistent with the inhibition of tRNA binding to the A-site during translation, tetracycline also inhibits the joining of releasing factors RF-1 and RF-2 during termination regardless of the stop codon. It is suggested that the binding of tetracycline on the second site i.e., at 16S rRNA on 30S subunit increases binding of non- or near-cognate tRNAs that would lead to misincorporation of wrong amino acids into the polypeptide chains as has been suggested for streptomycin. Pactamycin binds to two conserved nucleotides G693 and C795 that are universally conserved in the 16S rRNA of the small subunit and protects them from chemical modification and thereby reduces the flexibility of 30S subunit by locking two of the 16S rRNA helices 23 and 24 together and inhibits translocation but not initiation as predicted in earlier studies. Edeine is found to induce base-pairing between G693 and C795 conserved residues of 16S rRNA and inhibits tRNA binding to the P-site by preventing codon-anticodon interaction. Interestingly, pactamycin can rebreak the base pair formed by edeine. These findings with these two antibiotics antagonizing each other’s function highlights the importance of the G693:C795 base pair in 16S rRNA for the tRNA-binding at the P-site of the ribosome and its importance in translocation. Edeine is also found to act like streptomycin in promoting translational misreading. Both edeine and pactamycin are universal inhibitors. Puromycin that resembles the 3’ end of amino acylated tRNA can join the ribosomal A-site, accepts the growing polypeptide chain and then the next step of elongation is blocked. The growing polypeptide is released prematuredly. Puromycin affects both bacterial and eukaryotic translation. Kirromycin inhibits the conformational changes in ribosome that occur and facilitate the release of EF.Tu from ribosome after the hydrolysis of GTP. If the EF-Tu factor is not released then another EF-Tu carrying another aminoacylated tRNA cannot be delivered to the 70S complex. Fusidic acid prevents the release of EF-G.GDP from prokaryotic ribosome so that the next step in translation is arrested. Cycloheximide an inhibitor of peptidyl transferase activity is associated with the 60S subunit of eukaryotes. It prevents peptide bond formation. It blocks elongation and facilitates the formation of polyribosome complex. The polysome formation is a result of decreased elongation rather than increased initiation. 31 Chloramphenicol a prokaryotic translational inhibitor, reduces productive positioning of the aminoacylated tRNA at the A-site and thereby interferes in peptide bond formation. Sparsomycin an universal inhibitor of translation, promotes stacking interactions in the rRNA thereby promoting conformational alterations in the A- and P- sites of ribosome. Its primary action relies on the interaction with the P-site tRNA and it enhances the affinity of tRNA for the P-site (i.e., the tRNA movement is reduced from P to E-site) because of which translocation is blocked and protein synthesis is inhibited. Aminoglycosides (streptomycin, amikacin, tobramycin and neomycin) bind to the 30S subunit and block initiation of protein synthesis by forming aberrant initiation complexes. In addition, they cause miscoding at the aminoacyl-tRNA-mRNA step. Macrolides (erythromycin, clarithromycin and roxithromycin) containing a 14 or 15 or 16membered lactone ring, to which one or more deoxy sugars, usually cladinose and desosamine, are attached, bind to 23S ribosomal RNA at the peptidyl transferase center and act by producing a stearic blockage of the ribosome exit tunnel and hamper the progression of nascent chains. They generally bind to A2058, a ribosomal RNA nucleotide. Toxins produced by Diptheria and Pseudomonos modify elongation factor 2 in eukaryotes (eEF2) and inhibit translation. These toxins contain ADP-ribosyl transferase activity that transfers the ADP-ribose from NAD (nicotinamide adenine dinucleotide) to a modified histidine amino acid residue of the elongation factor 2 (EF2). ADP-ribosylated eEF2 the elongation step in protein synthesis. Ribosome inactivating proteins (RIPs) are plant proteins that inhibit of protein synthesis. Two types of RIPs are found. Type1 RIPs are N-glycosylated monomeric non-cytotoxic proteins. Type 2 RIPS are dimeric consisting of two different chains linked by a disulfide bridge. One of them is an enzymatic chain and the other is a lectin that recognizes membrane sugars, mostly galactose residues. Many type2 RIPs such as ricin, abrin, viscumin, modeccin and volkensin have been isolated to date. Ricin for example produced by castor seeds is a most potent among the toxins. Both types of RIPs have N-glycosidase activity and can act on the largest rRNA of the ribosomes. Interferons are cytokines with a particularly direct impact on protein synthesis in the virusinfected cells. They induce in turn enzymes like double-stranded RNA-dependent protein kinase (PKR) and 2’ 5’ oligo adenylate synthetase (2-5A Synthetase) that act at the cellular level as first line defenses against viral infection. Both these latent forms of enzymes are activated by doublestranded RNA through different mechanisms. In the presence of dsRNA, activated 2-5A synthetase produces a series of short 2’-5’ linked oligonucleotides pppA(pA)n, where n is generally 2 or 3. These short 2-5A oligonucleotides activate RNAse L, which degrades RNA mainly by cutting at the 3’side. The other enzyme PKR is a serine-threonine kinase and is also activated in the presence of dsRNA. Activated kinase phosphorylates the alpha subunit of eukaryotic initiation factor 2 (eIF2α) and inhibits the initiation of protein synthesis of general mRNAs. In fact phosphorylation of eIF2α is a stress signal and occurs in heme-deficiency, virusinfection, amino acid starvation and during accumulation of unfolded proteins through the activation of specific eIF2α kinases such as heme-regulated kinase (HRI), PKR, PERK (PKR32 like ER-resident kinase) and GCN2 (general control non-derepressible) respectively. In fact both HRI and PERK are activated in response to the accumulation of denatured proteins or unfolded proteins. Many times when the synthesis exceeds the folding, proteins cannot fold properly. Under such conditions, endoplasmic reticulum that is involved in the synthesis of secretory proteins evokes a complex signaling pathway called an unfolded protein response (UPR) that is primarily adaptive in nature. The UPR has two arms: translational attenuation that is mediated by the phosphorylation of the α-subunit of eIF2 and a transcriptional induction of chaperones that correct the protein folding. Translational regulation Translational control is defined as a change in the efficiency and utilization of mRNAs. The synthesis of different proteins is altered during development, differentiation, virus infection, hormonal treatment and depending on various external conditions. In many of these cases mentioned above, in addition to changes in the RNA synthesis (transcription), the changes in translational apparatus also regulate the synthesis of proteins. Very early experiments have shown that eggs contain mRNA that is not translated until fertilization. The rate of protein synthesis in these egg cells is shot up soon after fertilization. Until fertilization these mRNAs are masked with proteins (mRNP complexes) that keep the mRNAs silent. Deproteinized egg mRNA can be translated efficiently. In many animals, the first few hours of life after fertilization proceed with very little transcriptional activity. Maternal mRNAs present before fertilization play a crucial role in early decisions. Translational controls are important for establishing body axes i.e the anterior-posterior and dorsal and ventral-axes of the mature organisms. Similarly heme-deficiency has been shown to inhibit the translation of hemoglobin mRNA in reticulocytes, or in their lysates (cell extracts). Addition of hemin, the iron protoporphyrin compound, stimulates the hemoglobin synthesis in the cell-free translational systems derived from hemedeficient reticulocytes. Heat shock for example inhibits translation of normal mRNAs but facilitates the translation of heat shock mRNAs. Virus infection limits the synthesis of many host proteins but facilitate the synthesis of viral proteins. In most of these cases the regulation of protein synthesis occurs at the initiation level. However, protein synthesis is also found regulated at the elongation level. The best examples are the secretory proteins that are made on the surface of endoplasmic reticulum. The secretory proteins interact with a ribonucleoprotein complex called signal recognition particle (SRP) during the elongation phase. SRP is a ribonucleoprotein complex consisting of 7S RNA. The interaction of growing secretory polypeptide with SRP arrests the synthesis of the secretory polypeptide. The block in elongation is relieved in the presence of ER membrane protein called docking protein. The latter removes SRP bound to the growing polypeptide. This kind of a regulation appears to be essential for many of the digestive enzymes (most of them are secretory proteins) whose synthesis occurs only after a meal. When the enzyme is not needed the synthesis does not occur or it is arrested at the level of elongation. An analysis of these examples provided a wealth of information to understand the molecular mechanisms (role of different protein factors, small nuclear RNAs, covalent modifications of proteins, critical elements in mRNA structure) regulating translation in development, differentiation, health and disease. Reasons for translational regulation In spite of the fact that translation or protein synthesis is the last step in the overall gene expression, cells have evolved regulatory mechanisms that control overall translation as 33 mentioned above. These regulatory mechanisms affect the posttranscriptional modifications of mRNAs, transport of mRNAs to cytosol, assembly of amino acids, protein folding and degradation. It appears the cell can apply translational regulation much more easily because it takes very little time than regulating the transcription. Translational regulation is quite often more focused, fast and flexibile affecting the translation of selective messages or whole class of mRNAs as in heat shock and virus-infected host cells. Along with transcriptional regulation, translational controls offer to fine-tune the regulation of gene expression many times. In many systems like egg cells, reticulocytes and RNA viruses where there is limited transcriptional regulation, translational controls play most important role. This fact suggests that probably translational controls are in existence prior to the evolution of transcriptional controls. Regulation of cellular protein synthesis generates concentration gradients of proteins that affect the translation of other mRNAs or that determine the pattern formation during early development. Different RNAs and functions RNAs are generally single-stranded. However many RNAs fold back on themselves to form short regions of double helical structure that are complimentary to each other. As you will see many times bases in RNA do not follow strict Watson-Crick base pair rules as in DNA and there is some flexibility. For example the base uracil, which is specific to RNA, pairs with A and also sometimes with G. Stability of RNA is very poor compared to DNA, and is easily degraded. Unlike DNA, RNA has a ribose sugar with 2’OH that makes RNA easily susceptible for cleavage than the deoxyribose sugar present in DNA. Unlike DNA, different kinds of RNAs with different functions are produced. For example, RNA, like DNA, is the genetic material for many viruses. However, when these viruses infect their hosts, the RNA can be converted to DNA by an enzyme called reverse transcriptase and then the viral DNA can integrate with the host chromosomal DNA. Hence, like DNA that serves as a template for RNA synthesis, RNA also serves as a template for DNA synthesis in the presence of DNA polymerase-like enzyme called reverse transcriptase. In fact the RNA component associated with reverse transcriptase-like enzyme called telomerase is crucially required to replicate the 3’ends of the chromosomes. Telomerase is an RNA-protein complex. While telomerase plays an important role in the replication of the DNA at the ends of a chromosome, DNA polymerases play a role in the replication of the rest of chromosomal DNA. However DNA polymerases cannot initiate altogether the synthesis of a new DNA chain and can only elongate a given DNA chain from a 3’-OH group. To solve the DNA replication problems of both the leading and lagging strands, cells employ small RNA primers in DNA replication. In the splicing of RNAs, removal of the non-coding regions and joining of the coding regions in messenger RNAs is accomplished by small nuclear RNA-protein complexes that contain UI, U2, U4, U5 and U6 RNAs, and different proteins. While ribosomal RNA (rRNA) is an important component in the structure of ribosomes, the introns in some of the ribosomal RNA, also called as ribozymes act like protein enzymes in catalyzing their own cleavage from the pre-rRNA transcripts in the presence of guanosine as a cofactor, without the aid of any proteins. They not only have a nuclease activity (hydrolysis of RNA) but they also carry a polymerase activity and are able to join nucleotides through a process called transesterification reaction. 34 In fact one of the most important properties of the 16S ribosomal RNA associated with the small subunit includes the identification of the ‘start codon/site’ in the mRNA template and monitoring the codon-anticodon interaction. In contrast, the long rRNA transcripts of the large subunit plays a role in catalyzing the peptide bond formation between adjacent amino acids during the translocation step in protein synthesis. Thus some rRNAs like introns act on phosphorous centers and the long rRNA component (23S or 28S rRNA) of the large subunit is associated with the peptidyl transferase center can act on carbon centers during peptide bond formation. In the protein synthetic machinery, the messenger RNA (mRNA) template carries the information for the synthesis of proteins. The nucleotide sequences in mRNA however are identified by small adaptor molecules called transfer RNAs (tRNAs) that bring specific activated amino acids on to the template mRNA based on the sequence information in the mRNA. Interestingly, SsrA, kind of tmRNA (a combination of messenger RNA and transfer RNA with 457 nucleotides) plays an important role in the translation of broken mRNAs that do not have stop codons. When a stop codon is not found in broken mRNAs at the 3’ end, then SsrA RNA can be charged with alanine and binds to a stalled ribosome with the help of elongation factor EF-Tu and GTP. Then this alanine participates in the peptide bond formation of the incompletely made nascent polypeptide and ensures the release of the incomplete mRNA. Since SsrA RNA also contains a small open reading frame that codes for few amino acids and a stop codon, the incomplete polypeptide made from the broken mRNA is coupled with the small polypeptide by the SsrA RNA before it is released and the ribosome is recycled. Different RNAs or their structures play a role in the regulation of gene expression both at the transcription and translational levels. For example, binding of the signal recognition particle (SRP) an RNA protein complex, consisting of 7S RNA, to an mRNA that codes for secretory protein synthesis on the surface of endoplasmic reticulum, halts the synthesis of the secretory protein and regulates the translational elongation. There are several examples where in formation of hairpin-like structures in mRNAs can regulate gene expression at transcriptional and translational level. The structures in RNA serve as sensors for metabolites and uncharged tRNAs. These RNA regulatory elements are called riboswitches. Another form of RNA called the guide RNA, has an ability to add or delete a ribonucleotide in an mRNA and can edit RNAs. RNA editing is a different way to produce different proteins from an mRNA sequence. In addition to these different forms and functions of RNAs, recent studies lead to the discovery of micro RNAs (miRNAs) and silencing or small-interfering RNAs (siRNAs) that are complimentary to certain regions of an mRNA. Because of this complimentarity, these RNAs can base-pair and inhibit the translation or degrade the mRNAs. MicroRNAs are transcribed in the nuclei and are processed by RNAse III –like enzyme. The precursor 70 nt miRNAs then folds into stem-loop structures and are transported to the cytoplasm where they are further processed to yield mature miRNAs of ~20 nt duplex intermediate by an RNAase-III enzyme called Dicer. Dicer also processes long double stranded RNA-precursors to produce siRNAs. The processed miRNAs and siRNAs assemble into different complexes containing various proteins called miRNP (microRNA protein complex) and RISC (RNA-induced silencing complex) that guide them to the respective mRNAs where the miRNAs or siRNAs base-pair to the complementary regions in mRNAs and inhibit the translation or degrade the mRNAs respectively. The only common protein of both the complexes is Argoanuate and the functions of these proteins are not clear. Further small double-stranded RNA can also activate protein kinases like PKR that 35 phosphorylate the alpha –subunit of translational initiation factor 2 (eIF2α) and also 2’-5’ A synthetase that produce 2-5A oligonucleotides which activate RNAse L that degrades RNA as mentioned under the subheading of interferons. Many such examples raise the question whether RNA is evolved prior to DNA? Co-and post-translational modifications of proteins Proteins, and/or their amino acids, undergo covalent modifications during (co-translational) or after (post-translational) their synthesis. These modifications play critical role in the synthesis, stability, structure, function, degradation, and in targeting the proteins to different cellular locations. The post translational modifications also influence gene expression, and the interaction of these proteins with other proteins. The covalent modifications of proteins signal the development, differentiation, degradation, and cell death. Post translational modifications of proteins can arise due to the addition of certain functional groups to certain amino acids, addition of a protein or peptide, a change in the chemical nature of amino acid, and protein-processing that is required for attaining a proper structure or conformation of the protein. a) Modifications of some of the amino acids: In modifications such as glycosylation, phosphorylation, ADP-ribosylation, iodination, sulfation, acetylation, alkylation and formylation, addition of some of the functional groups like sugars, phosphate, ADPribose, methyl, iodine, sulfur, acetyl, methyl, formyl groups respectively can modify various amino acids in proteins and their functions. b) Addition of a protein/ peptides, amino acids, or lipids: Examples here include the addition of ubiquitin protein through an isopeptide bond (as in ubiquitination, see below) or a ubiquitin-like peptide (SUMOylation); addition of an amino acid such as polyglutamic acid residues to the C-terminus of proteins (as in glutamylation), and addition of lipids such as myristic acid (myristoylation) at the N-terminal end, or addition of prenyl group to the cysteines located near the C-terminal end, and addition of palmitate to a cysteine at N or C-terminus or in the body of the protein. c) Change in the chemical nature of amino acids: Conversion of one amino acid to another as in the case of citrulination, or deamidation where arginine is converted to citruline or racemization that converts one optical isoform of an amino acid to another as in the conversion of L-amino acid to D-amino acid d) Structural changes in proteins that involve addition of S-S- bridges between cysteine residues and enzymatic or non-enzymatic cleavage of peptide bonds. A protein may undergo more than one modification (like processing or removal of some amino acids and addition of disulfides as in insulin). Some of them are reversible and some of them are irreversible. Phosphorylation Phosphorylation is a reversible modification. Addition of a phosphate moiety to the hydroxyl groups in serine, threonine and tyrosine amino acids can occur in many proteins by respective kinases. Dephosphorylation is facilitated by protein phosphatases. The conversion of glucose monomers to the glycogen polymer requires the enzyme glycogen synthase in vertebrates. Phosphorylation of glycogen synthase generates inactive form of the enzyme. Dephosphorylated 36 form generated by a protein phosphatase is found to be active. Many factors involved in the initiation and elongation of eukaryotic translation (protein synthesis) and transcription (RNA synthesis) regulate their activity through phosphorylation-dephosphorylation. For example phosphorylation of a small portion of the alpha-subunit of heterotrimeric eukaryotic initiation factor 2 (eIF2α) that occurs in response to diverse stress conditions inhibits general protein synthesis but upregulates the translation of certain messenger RNAs like ATF4 or GCN4. It is a stress signal and affects stress-induced gene expression. Phosphorylation of the serine 51 residue in the alpha-subunit of heterotrimeric eIF2 affects its interaction with other proteins. For example, it forms a complex with another rate-limiting pentameric initiation factor called eIF2B and inhibits the ability of eIF2B to recycle inactive eIF2.GDP to eIF2.GTP. Methylation and Acetylation The N-terminal tails of H3 and H4 histone proteins (proteins that wrap around the chromosomes) undergo several reversible modifications such as phosphorylation (as described above), acetylation and methylation. Methylation occurs on lysine or arginine residues whereas acetylation occurs on lysine residues. All of them are reversible modifications. Acetyl transferases and methyl transferases are the enzymes that catalyze the addition of an acetyl group to a lysine, and a methyl group to arginine or lysine respectively. Histone acetylation has a role in chromatin assembly and transcription. Acetylated histone proteins are found associated with regions of chromosomes that are transcriptionally active whereas deacetylated proteins are found associated with inactive chromosomes thereby suggesting acetylation of histone proteins may regulate RNA synthesis. In contrast, methylated histone proteins are found associated with both transcriptionally active and repressed chromosomes. It is likely the function varies depending on the position and the amino acid that is modified. Glycation and Glycosylation Addition of one or several carbohydrate groups occurs in most of the secretory proteins that are synthesized on the surface of endoplasmic reticulum and are eventually found on the cell surface of eukaryotes. Generally the cytoplasmic proteins are not glycosylated except in pathological conditions like diabetes where the blood glucose levels are high. For example, glycosylation of hemoglobin that occurs non- enzymatically between the C-1 carbon of glucose and the Nterminus of hemoglobin polypeptides is taken as a measure to monitor the severity of diabetes. Glycation or addition of a glucose non- enzymatically differs from glycosylation. In glycation, the free amino group in proteins reacts with glucose forming a ketoamine called Amadori product. The glycated proteins forms cross links with other proteins, leading to structural and functional modifications. This modification is generally observed in collagen, lens crystalline and basement membrane proteins during ageing. In contrast, glycosylation of normal proteins is an enzyme-catalyzed process. Two classes of glycosylations are identified. These are O-linked and N-linked. In O-linked glycosylation, the sugars are added to serine or threonine amino acids generally and also to a modified hydroxylysine as in the case of collagen protein. O-linked glycans are present in the blood group antigens. In N-linked glycosylation, sugars are added to the amide nitrogen of aspargine. In addition to these differences, O-linked glycosylation appears to be a post-translational event and occurs after the synthesis of the protein. In contrast, N-linked glycosylation is a co-translational event and occurs during the synthesis of proteins and mainly on the endoplasmic reticulum. 37 Further, the mechanisms involving the transfer of sugars to the corresponding proteins in the Oand N-linked glycosylations differ. Attachment of sugars to the proteins requires energy input. In both cases, the sugar is first complexed with a nucleotide (ex; UDP-glucose or galactose or UMP-glucose; GDP-mannose etc.,) generally in cytosol. But CMP-sialic acid is formed in the nucleus. In O-linked glycosylation, the activated sugar, or the nucleotide-linked sugar, is then transferred to a serine or threonine amino acid of a protein by a membrane bound glucosyl transferase enzyme. Each type of sugar transfer to a protein is catalyzed by a different type of glycosyl transferase. Glucosyl transferase enzymes not only use nucleotide- linked sugars but also use lipid- linked sugars and transfer them to the corresponding proteins as in the case of Nlinked glycosylation. N-linked glycosylation is a complex process. Here, the nucleotide sugar is first transferred to a lipid intermediate called dolichol phosphate by a glucosyl transferase enzyme before it is transferred to the amide nitrogen of aspargine of a glycosylated protein that is placed in a sequence Asn-X (any amino acid)-Ser/thr-Protein by an ologosachharyl transferase. However, some of these residues are processed or removed when the proteins are processed in the lumen of the endoplasmic reticulum or by glycosidases. An analysis of oligosaccharides of N-linked-glycosylated proteins reveals however that mostly they all contain a core pentasaccharide structure that is generally (3 mannose and two N-acetylgucosamine residues). The first sugar N-acetylglucosamine is the one linked to the aspargine residue of the protein. In addition to the core oligosaccharide residues, many N-linked glycosylated proteins have other sugar residues added to the protein. Further, some of these sugar residues (like the mannose residues linked to asparagines) of secretory proteins that are destined to reach lysosomes are also found phosphorylated. Examples of N-linked glycosylation include ribonuclease B, human IgG. Glycosylation plays a role in providing a correct charge, increases the solubility, stability and conformation of proteins. Different types of glycosylation regulate the targeting of proteins to different cellular locations or organelles. Oxidation-reduction Many of the cytoplasmic proteins in higher eukaryotes maintain their –SH groups because of a higher reducing potential in the cytoplasm and may not use these –SH groups to form disulfide bonds as a means to stabilize their structure. In contrast, several secretory proteins like immunoglobulins and hormones (like insulin) or digestive enzymes carry disulfide bonds that are formed between two cysteine residues (Cys-S-S-Cys) during or soon after their synthesis. These disulfide bridges are required to maintain the secondary structure of the protein. Oxidative modification of proteins can also occur by oxygen free radicals, metal-catalyzed oxidation systems and mixed function oxidation systems. These systems can oxidize some amino acids like proline, arginine and lysine to carbonyl derivatives. The amount of carbonyl content of proteins has been used to estimate protein oxidation during ageing. ADP-ribosylation It involves the addition of ADP-ribosyl moiety to an arginine or modified histidine amino acid residue in the presence of an enzyme ADP-ribosyl transferase. The ADP-ribosyl moiety comes from NAD+. A modified histidine residue in elongation factor 2 (EF2) in eukaryotes can be ADP-ribosylated in the presence of diphtheria toxin that contains ADP-ribosyl transferase activity. ADP-ribosylated EF2 inhibits protein synthesis. Cholera toxin also contains similar 38 enzymatic activity and is used to demonstrate that ADP ribosylation of the Gα subunit of the trimeric G-protein can block the GTPase activity associated with the protein and can convert the α-subunit into an inrreversible activator of adenylate cyclase, the enzyme that produces cyclic AMP from ATP. Iodination The synthesis of thyroid hormones, mainly thyroxine (T4) and triiodothyronine (T3) by thyroid gland, involves iodination of the tyrosine ring. The iodide ion required for this process is obtained from blood serum. Deficiency of iodine results in goiter, a disease in which thyroid gland grows abnormally. Thyroid hormones affect the expression of several genes that are involved in various metabolic processes. Formylation Formylated methionine is the first amino acid to be incorporated in all the bacterial proteins. Transformylase enzyme modifies methionine that is already attached to the initiator tRNA in prokaryotes by transferring a formyl group from N-10 formyl tetrahydrofolate. Deformylase enzyme removes the formyl group during the elongation step of protein synthesis. An amino peptidase enzyme also removes the first methionine from many proteins. Formylation favors selection of formylated Met-tRNAfMet by IF2 protein factor in prokaryotes, blocks the joining of the f-Met-tRNAfMet to the elongation factor EF-Tu. Lipoylation This refers to proteins with covalently attached lipids such as myristic acid, a fourteen –carbon saturated fatty acid (C14: 0), palmitic acid, a saturated sixteen –carbon fatty acid (C16: 0), farnesyl, fifteen carbon isoprenoid, and geranylgeranyl, a twenty carbon isoprenoid etc., The modifications are called as N-myristoylation, palmotoylation and prenylation respectively. In Nmyristoylation, myristc acd is bound to the N-terminal glycine of a variety of proteins by an amide bond. Myristoyl-CoA:protein:N-myristoyl transferase enzyme acylates the nascent polypeptides after cellular methionyl aminopeptidases remove the first or initiation methionine. Substrates for myristoylation include kinases like protein kinaseA, phosphatases like calcineurin B, transmembrane proteins like the alpha-subunit of heterotrimeric G-protein and a retroviral polyprotein gag coded by HIV. Prenylation involves the attachment of a prenyl group (farnesyl diphosphate or geranylgeranyl diphosphate) to the cysteines located near the C-terminus of a protein by a thioester bond whereas palimitoylation involves the addition of a palimitate to a cysteine by protein palimitoyl transferase to the N- or C-terminus of the protein and can also occur in the body of the protein. Most Ras proteins are substrates for palmitoylation. The γsubunit of G-protein coupled receptor in the retina is a substrate for prenylation. Palmitoylation can also occur at times nonenzymatically. Palimitoylation increases the hydrophobicity of a protein molecule so that its association with cellular membranes can occur whereas simple myristoylation or prenylation is not sufficient for a protein to interact with the plasmamebrane. Addition of another lipid molecule in the case of myristoylation or methylation of the carboxyl groups of cysteine in prenylation may enhance their affinity for membranes. 39 Protein Splicing Like RNA splicing and maturation, most proteins produced soon after their synthesis lose one or several of the amino acids to form a mature and functional protein with a proper structure. The removal of the amino acids in proteins can occur in the presence and absence of enzymes. Many proteins contain sequences called inteins that have an endonuclease domain inserted into the protein and exteins. Inteins are excised in the final protein and the mature host protein contains exteins. The inteins are comparable to introns and the exteins are to exons in RNA. The removal of inteins is autocatalytic and does not involve any enzymes. Most inteins have a central conserved aminoacid sequence (LAGLIDADG) of an endonuclease domain and many inteins also do not have such an endonuclease domain. The DNA sequences responsible for protein inteins can be modified so that the protein splicing can be controlled at the will of the researcher. Protein processing Protein processing can also occur by different proteases. Processing facilitates the generation of biologically active protein. Many of the digestive enzymes and hormones are synthesized as prepro proteins containing several additional amino acids that are not found in the mature and processed protein. These additional amino acids are removed by proteases. Examples include many of the pancreatic proteases like trypsin, elastase, enteropeptidase and carboxypeptidase. Most of these enzymes are made on the rough surface of endoplasmic reticulum and are then moved into the lumen of the endoplasmic reticulum. Here the N-terminal signal sequence characteristic of the secretory proteins is cleaved by a signal peptidase. Afterwards, some of these proteins are further processed. For example the conversion of chymotrypsinogen to active π chymotrypsin is done by trypsin that cleaves peptide between arginine 15 and isoleucine 16 at the N-terminus of the protein. This is further followed by autocatalytic processing of πchymotrypsin that removes amino acid residues 14-15 and 47-148 from the molecule to produce final α-chymotrypsin. The cleavage of a hexapeptide from the N-terminus end of trypsinogen in duodenal cells to active trypsin occurs by enteropeptidase. Active trypsin also catalyzes inactive trypsinogen to active trypsin. Another important example involves a cascade of proteolytic activation of specific proteases that will process prothrombin to thrombin which in turn catalyzes the conversion of fibrinogen, a serine protease, to fibrin during blood clotting. Thrombin cleaves mainly a bond between Arg-Gly. Similarly in programmed cell death, cysteinyl aspartate proteases called caspases get activated and act on procaspases and convert them to active caspases. The proteases identify specific amino acid sequences in proteins. Active caspases in turn cleave several protein substrates and modify their activities. Other modifications Other covalent modifications include alkylation (addition of methyl or ethyl groups to lysine or arginine amino acids), glutamylation (covalent linkage of glutamic acid residues to the cterminus of tubulin) and sulfation, addition of sulfate group to tyrosine). Further, often there is an interconversion of optical isoforms of amino acids (L-amino acid to D-amino acid) called racemization and conversion of a neutral amide group to an acidic group by deamidation. Spontaneous deamidation of the amino acids of glutamine and aspargine occurs in several proteins during ageing. 40 Ubiquitnation Many proteins, prior to degradation by a proteaosome, are coupled or conjugated enzymatically with a protein called ubiquitin (Ub). Ubiquitin tag to a protein signals for degradation. This type of covalent modification is different from other modifications of amino acids that have been described above. Here the target or substrate protein is attached with another polypeptide like ubiquitin. Recent studies indicate that many small polypeptides that are distinct from ubiquitin but related to ubiquitin are tagged enzymatically to target molecules in different biological processes such as DNA repair, autophagy and signal transduction. Ubiquitination of proteins provides large and better chemical surface. The process requires three to four enzymes. The sequence of events that occur during ubiquitination are as follows: In step 1, a deubiquitinating enzyme processes the precursor ubiquitin protein to generate ubiquitin that contains Gly-Gly sequence at the C–terminus. In step 2, activation of ubiquitin occurs by enzyme 1 and ATP. In the activation step, C-terminus of ubiquitin is adenylated by enzyme 1. Adenylated ubquitin is then linked to the enzyme through a high-energy thioester bond. In step 3, activated ubiquitin is passed to cysteinyl group of the enzyme 2 called ubiquitin-conjugating enzyme (Fig. 19). In some cases, enzyme 3, ubiquitin-protein ligase, combines ubiquitin and the target protein (substrate) through an amide (isopeptide) bond. Otherwise enzyme2-conjugated ubiquitin is sufficient for modification of the substrate protein in many cases. Ubiquitin (Ub) is covalently attached to other proteins (target protein) through an isopeptide bond. The isopeptide bond occurs between the carboxy-terminal glycine of Ub and the epsilonamino group of lysine in the target protein (Fig. 19). Target protein could be another ub, or ubiquitin-like protein (SUMO or small Ub-like modifier, autophagy-12, interferon stimulated gene of 15 kDa) or structurally a different protein substrate. Depending on the number of ubiquitin molecules attached, the function may differ. For example, mono- and di- ubiquitination are implicated in endocytosis, and a chain containing minimum four molecules of Ub may be required for efficient proteasomal degradation. Also the lysine residues in Ub that is involved in the linkage or in the formation of isopeptide bond may play a role. Lysine (K) 48 –linked chains are involved in proteasomal degradation and K63 linkage may be involved other processes like endocytosis or DNA-repair. Fig. 19: Ubiquitination 41 Suggested Readings 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. J. Darnell, H. Lodish, D. 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