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tRNA is a link between the mRNA and the polypeptide being synthesized During translation proteins are synthesized according to a genetic message of sequential codons along mRNA Translation Transfer RNA (tRNA) tRNA)– is an interpreter between the two forms of information – base sequence in mRNA and aminoacid sequence in polypeptides tRNA aligns appropriate aminoacids to form a new polypeptide. To perform this action tRNA must: •Transfer amino acids from the cytoplasm’s amino acid pool to a ribosome •Recognize the correct codons in mRNA tRNA – an adaptor molecule Molecules of tRNA are specific for only one particular amino acid Each type of tRNA associates a distinct mRNA codon with one of the 20 amino acids used to make proteins •One end of tRNA molecule attaches to a specific amino acid •The other end attaches to an mRNA codon by base-pairing with its anticodon. Anticodon – a nucleotide triplet in tRNA that base pairs with a complementary codon in mRNA. tRNAs decode the genetic message, codon by codon. Transfer RNA's (tRNA) Function: carries amino acids to the ribosome for assembly into polypeptides. Therefore: translates the mRNA genetic code. Mechanism tRNA Structure •complementary and antiparallel base pairing of the anticodon on the tRNA molecule paires with the mRNA codon. •determines which amino acid is added by the ribosome to the growing polypeptide. Structure •Single strand of RNA, 80 bp long •Folded into clover leaf configuration, driven by complementary base pairing. •Anticodon loop •Amino acid-binding site (3'-end) 1 All tRNAs have a similar structure The shortest functional RNAs know: the smallest are 74 nt in length, the largest – ralely more than 90 nt. They were among the first RNAs discovered Clover leaf: Acceptor arm, formed by 7bp between the 5’and 3’ ends of the molecule The aa attaches at the extreme 3’ end of the tRNA, to the adenosine of the invariant of terminal CCA sequence The D arm – named after modified nucleoside dihydrouridine, which is always present in this structure The anticodon arm, containing triplet called anticodon that will base-pair with mRNA during translation The V-loop, which contains 3-5 nts or 13-21 nts. The TΨC arm, named after the sequence of thymidinepseudouridine-cytosine, which it always contains An aminoacyl-tRNA synthetase joins a specific amino acid to a tRNA. Linkage of the tRNA and amino acid is an endergonic process that occurs at the expense of ATP. AminoacylAminoacyl-tRNA synthetase – enzyme that catalyzes the attachment of an amino acid to its tRNA The correct linkage between tRNA and its designated amino acid must occur before the anticodon pairs with its complementary mRNA codon. Each of the 20 amino acids has a specific aminoacyl-tRNA synthetase The proper synthetase attaches an amino acid in two steps. ATP is needed. 1. Activation of the amino acid with AMP. The synthetase’s active site binds the amino acid and ATP; the ATP loses two phosphate groups and attaches to the amino acid as AMP. 2. Attachment of the amino acid to RNA. The appropriate tRNA covalently bonds to the amino acid, displacing AMP from the enzyme’s active site. The aminoacyl-tRNA complex releases from the enzyme and transfers its amino acid to a growing polypeptide on the ribosome. AminoacylAminoacyl-tRNA synthetase activity 1. The active site of the enzyme binds the amino acid and an ATP molecule. 2. The ATP loses two phosphate groups and joins to the amino acid as AMP (adenosine monophosphate). 3. The appropriate tRNA covalently bonds to the amino acid, displacing the AMP from the enzyme's active site. 4. The enzyme releases the aminoacyl tRNA, also called an "activated amino acid." 2 AminoacylAminoacyl-tRNA synthetase has affinity to its tRNA This is due to the extensive interaction between the two, covering 25nm2 of the surface area and involving the acceptor arm and the anticodon loop of the tRNA, as well as individual nucleotides in the D and the TΨC arm The interaction between enzyme and aa is less extensive, the aa is smaller, several pairs of aa are structurally similar. Errors do occur, but at a low rate. Genetic code – the set of three-base code words (codons) in mRNA that stand for the 20 aa in proteins. •Code is non-overlapping - each base is a part of only one codon •Code is devoid of gaps, or commas -each base is a part of codon. Frameshift mutations When the enzyme attaches the wrong aa to a tRNA, this aa is subsequently transformed to correct by a separate reaction. This was first discovered in B. megaterium. Glutamic acid – glutamine conversion by transamidation reaction. Asparagine-tRNA from aspartic acid-tRNA in other bacteria (not E. coli) Breaking the genetic code It was broken by using synthetic messengers of synthetic trinucleotides and observing the polypeptides synthesized or aminoacyl-tRNAs bound to ribosomes. Code is degenerate: •There are 64 codons in all. •Three are stop-codons •The rest code for amino acids. The genetic code There are only about 45 distinct types of tRNA. This is enough to translate 64 codons, as some tRNA recognize two or three codons specifying the same amino acid. This is possible because the rules are relaxed between the third base of an mRNA codon and the corresponding base of a tRNA anticodon. This exception to the base-pairing rule is called wobble. 3 Ribosomes Ribosomes coordinate the pairing of tRNA anticodons to mRNA codons Ribosomes have two subunits – large and small – separated when not involved in protein synthesis. Ribosomes are composed of about 60% ribosomal RNA (rRNA) and 40% protein. Ribosomes In addition to an mRNA binding site, each ribosome has three tRNA binding sites (P, A and E). •The P site holds the tRNA carrying the growing polypeptide chain •The A site holds the tRNA carrying the next amino acid to be added •Discharged tRNAs exit the ribosome from the E site. site Ribosomes Large and small subunits of the ribosomes are •Constructed in the nucleolus •Dispatched through nuclear pores to cytoplasm •Once in the cytoplasm, are assembled into functional ribosomes only when attached to an mRNA Compared to eukaryotic ribosomes, prokaryotic ribosomes are smaller and have a different molecular composition. The anatomy of a ribosome. (a) A functional ribosome consists of two subunits, each an aggregate of ribosomal RNA and many proteins. (b) A ribosome has an mRNAbinding site and three tRNAbinding sites, known as the P, A, and E sites. (c) A tRNA fits into a binding site when its anticodon base-pairs with an mRNA codon. The P site holds the tRNA attached to the growing polypeptide. The A site holds the tRNA carrying the next amino acid to be added to the polypeptide chain. Discharged tRNA leaves via the E site. 4 Ribosomes Ribosome structure Microscopy led to initial progress in understanding structure of ribosomes. In 1940s – first photo-micrographs of bacterial ribosomes - oval – structures, 29 x 21 nm Eukaryotic ones – bigger, about 32 x 22 nm Ultra centrifugation was used to measure sizes of ribosomes and their composition. Each ribosome has two subunits: In eukaryotes – 60S and 40S In bacteria – 50S and 30S NB: sedimentation coefficients are not additive because they depend upon shape as well as upon mass: it is perfectly acceptable for the intact ribosomes to have the S coefficient less than a sum of subunits. Sedimentation coefficients of intact eukaryotic ribosomes – 80S Prokaryotic – 70S. They can be broken into smaller components. Ribosomes The large subunit contains three rRNAs in eukaryotes: 28S, 5.8S and 5S rRNA Steps of translation In bacteria – only rRNAs two 23S and 5S rRNA. In bacteria the equivalent of the eukaryotic 5.8S rRNA is contained within the 23S rRNA. Building of a polypeptide, or translation occurs in three stages: The small subunit contains a single rRNA in both types of organisms: an 18S rRNA in eukaryotes and a 16S rRNA in bacteria. 1. Initiation Both subunits are associated with a variety of ribosomal proteins: Eukaryotes Bacteria 60S – 50 proteins 50S – 34 proteins 40S – 33 proteins 30S – 21 proteins 2. Elongation 3. Termination The proteins of the small subunits are called S1, S2 etc.; those of the large one – L1, L2, etc. There is one of the proteins per each ribosome, except for L7 and L12, which are present as dimers. 5 Initiation Initiation brings together mRNA, a tRNA attached to the first amino acid (aa) (initiator tRNA, the 1st aa is always methionine), and the two ribosomal subunits. Initiation in bacteria The main difference between initiation of translation in bacteria and eukaryotes is that in bacteria the translation initiation complex is built up directly over the initiation codon, the point at which protein synthesis will begin. Eukaryotes, use a more indirect process for locating the initiation point. In bacteria, the process initiates when a small subunit, in conjunction with the translation initiation factor IF-3, attaches to the ribosome binding site –Shine-Dalgarno sequence. Shine-Dalgarno sequence – a short target site, consensus sequence 5’-AGGAGGU-3’ in E. coli, located about 3-10 nucleotides upstream the initiation codon, where the translation begins. Initiation in bacteria The ribosome binding site is complementary to the region at the 3’-end of the 16S rRNA, the one present in the small subunit. This base pairing is involved in the attachment of the small subunit to the mRNA. Attachment positions the small subunit over the initiation codon. This codon is usually 5’-AUG’3’, but sometimes may be 5’GUG-3’, or 5’-UUG-3’. All three codons are recognized by the same initiator tRNA, the last two by wobble. Initiation of translation in prokaryotes •The modification attaches a formyl group, -COH, to the amino-group which means that only the carboxyl group of the initiator methionine is free to participate in peptide bond formation. •This ensures that polypeptide bond synthesis can take place only in the N→C direction. •The initiator tRNA Met is brought to the small subunit of the ribosome by a second initiation factor, IF-2, along with the molecule of GTP, the latter acts as energy source. •Only tRNA Met is only able to decode initiation codon. •NB: During elongation the internal AUG codons are recognized by different tRNA Met, carrying unmodified methionine. •Completion of initiation phase: when IF-1 binds to and stabilizes initiation complex, enabling the large subunit to attach. •Attachment of the large subunit requires energy. 6 Summary of prokaryotic translation initiation 1. Dissociation of 70S into 50S and 30S subunits, under the influence of IF-1. 2. Binding of IF-3 to the 30S subunit, which permits reassociation between the ribosomal subunits. 3. Binding of IF-1, IF-2 and GTP alongside IF-3. Summary of prokaryotic translation initiation 4. Binding of mRNA and tRNAi Met to form the 30S initiation complex. These components can bind in either order, but IF-2 sponsors tRNAi Met binding, and IF-3 sponsors mRNA binding. In each case the other factors also help. 5. Binding of 50S subunit, with the loss of IF-a and IF-3. 6. Dissociation of IF-2 from the complex, with simultaneous hydrolysis of GTP. The product is the 70S initiation complex, ready to begin elongation. Initiation in eukaryotes is mediated by the cap structure and poly(A)tail Simplified version of the scanning model for translation initiation The 40S ribosomal subunit, alongside with factors, tRNAi Met and GTP recognize the m7G cap at the 5’-end of an mRNA and allow the ribosomal subunit to bind at the end of the mRNA. The 40S subunit is scanning the mRNA toward the 3’-end, searching for the initiation codon, melting the stem loop structure in its way., The ribosomal subunit locates an AUG initiation codon and stops scanning. Now the 60S ribosomal subunit can join the complex and initiation can occur. Initiation in eukaryotes Only small number of eukaryotic mRNAs have internal ribosome binding sites. The small subunit of the ribosome makes its initial attachment at the 5’-end of the mRNA and scans along the RNA sequence to find the AUG. The first step involves the pre-initiation complex: 40S subunit of the ribosome, GTP, tRNAi Met and eukaryotic factor eIF-2 (a trimer of three different proteins). After assembly the pre-initiation complex associates with the 5’end of mRNA. This step requires the cap binding complex. 7 Initiation in eukaryotes Initiation in eukaryotes After attachment to 5’end the complex is called initiation complex. The attachment is also influenced by polyA. This is thought to be mediated by polyadenylate-binding protein (PADP) which is attached to polyA. It can scan the RNA. PADP forms association with eIF-4G, requiring the mRNA bends back on itself. eIF-4A and eIF-4B: eIF-4A and possible eIF-4B have helicase activity and are able to break intramolecular base-pairs in mRNA freeing the passage for ribosome. With artificially uncapped DNA the PADP interaction is sufficient to load pre-initiation complex. The initiation codon, the 5’-AUG-3’ is within the consensus sequence – 5’-ACCAUGG-3’, called Kozak sequence. Leader sequences in eukaryotes are long – up to 100 or more bp, have structures – hairpins and other. When initiation complex is positioned over the initiation codon, the large subunit attaches. The elongation cycle of translation - overview Elongation is similar in eukaryotes and prokaryotes Several proteins called elongation factors take part in this threestep cycle which adds amino acids one by one to the initial amino acid: 1. Codon recognition. 2. Peptide bond formation. 3. Translocation. Not shown are the elongation factors and GTP. 8 Elongation 1. Codon recognition. • The mRNA codon in the A site of the ribosome forms hydrogen bonds with the anticodon of an entering tRNA carrying the next amino acid in the chain. • An elongation factor EF-Tu directs tRNA into the A site in bacteria. In eukaryotes – eEF-1 (4 subunits: eEF-1α, eEF-1β, eEF-1γ, eEF-1δ) • eEF-1α consists of eEF-1α1 and eEF-1α2 • Hydrolysis of GTP provides energy for this step. 2. Peptide bond formation. •A peptide bond is formed between the polypeptide in the P site and the new amino acid in the A site by a peptidyl transferase. This reaction requires hydrolysis of GTP bound to EF-Tu, or eEF-1. •This inactivates EF-Tu, it is ejected from the ribosome and regenerated by EF-Ts. No eukaryotic homology of EF-Ts is known, but possibly one of the subunits of the eEF-1 has such activity. •Peptidyl transferase activity appears to be one of the rRNAs in the large ribosomal subunit . •The polypeptide separates from its tRNA and is transferred to the new amino acid carried by the tRNA in the A site. Elongation Elongation of translation 1. 3. Translocation. •The tRNA in the A site, which is now attached to the growing peptide, is translocated to the P site. Simultaneously, the tRNA that was in the P site is translocated to the E site and from there it exits the ribosome. •During this process, the codon and anticodon remain bonded, so that mRNA and the tRNA move as a unit, bringing the next codon to be translated into the A site. •The mRNA is moved through the ribosome only in the 5’ to 3’ direction. •Translocation requires GTP hydrolysis and is mediated by EF-G in bacteria and by eEF-2 in eukaryotes. 2. EF-Tu with GTP binds amino-acyl tRNA to the A site. Peptidyl transferase forms bond between peptide in the P site and the newly arrived amino-acyl tRNA in the Asite. This lengthens peptide and shifts it to the A site. 9 Elongation of translation 3. EF-G, with GTP, translocates the growing peptidyl tRNA, with its mRNA codon to the P site. Termination of translation 3. Eukaryotes have just one factor - eRF. 4. In bacteria – process is energy-independent. 5. In eukaryotes – requires hydrolysis of GTP. 6. Termination results in release of completed polypeptide from tRNA in the P site, and dissociation of the translation complex. 7. Ribosome subunits enter the cytoplasmic pool where they remain until used again in another round of translation. Termination of translation 1. A site is entered not by tRNA but by a protein called release factor. The release factor hydrolyzes the bond between the tRNA in the P site and the last amino acid of the polypeptide chain. The polypeptide is thus freed from the ribosome. 2. Bacteria have 3 of release factors: RF-1 which recognizes the termination codons UAA and UAG RF-2 which recognizes UAA and UGA RF-3 which plays supporting role. Post-translational processing of proteins Translation is not the end of the gene expression. The polypeptide that emerges is inactive, but before it must undergo at least the first of the following steps: Protein folding. The polypeptide is inactive until it is folded into its correct tertiary structure. In cells folding is aide by molecular chaperones. In E. coli, the chaperones are divided into 2 groups: The Hsp70 chaperones, which include proteins called Hsp70,coded by dnaK gene, and Hsp40 coded by dnaJ gene and the GrpE. The chaperons bind to hydrophobic regions of the proteins, including proteins that still are being translated. They prevent aggregation by holding the protein in an open confirmation until it is completely synthesized and ready to fold. They also are involved in other processes that require shielding of hydrophobic regions. The chaperonins, the main version of which is E. coli is GroEL/GroEs complex. The complex is a multisubunit structure that looks like a hollowed-out bullet. Protein enters unfolded and exists folded. Mechanism - unknown 10 Post-translational processing of proteins Eukaryotic folding Proteins equivalent to chaperons and chaperonins have been identified. Eukaryotic folding makes less use of chaperonins, and more depends upon the action of Hsp70 chaperons. Post-translational processing of proteins Proteolytic cleavage. Some proteins are processed by cutting events carried out by enzymes called proteases. These cutting events may remove segments from one or both ends of the polypeptide, resulting in a shortened form of a protein, or they may ct polypeptide into a number of different segments, each one of which is active Chemical modification. Individual amino acids in polypeptide might be modified by attachment of new chemical groups. Intein splicing. Inteins are intervening sequences in some proteins, similar in a way to introns in mRNAs. They have to be removed and the exteins ligated in order for the protein to become active. Reading: Chapters 17, 18. References: R. Weaver, Molecular Biology, 2005. T. A. Brown, Genomes, 1999. 11