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SAN DIEGO MESA COLLEGE SCHOOL OF NATURAL SCIENCES General Biology Lecture (BIOL107); Instructor: Elmar Schmid, Ph.D. Chapter 10: DNA transcription, protein synthesis & The genetic code - Part II - “ DNA transcription and ribosomal protein translation are the crucial cellular processes which guarantee the controlled flow of genetic information from the digital molecular code of DNA to the final cellular protein product.” the information for the shape and function of the cell’s and organism’s proteins and enzymes is laid down in a molecular (or genetic) code contained in the DNA molecule in form of coded units called genes the scientific study of the molecular gene reading and genetic code-translating process occurring in cells of all biological organisms on planet Earth lead to the discovery of two of the most fascinating and elementary biological processes – DNA transcription and protein translation our modern understanding of these two biological processes came a long and difficult way in human history H Hiissttoorryy 1909: the English physician A. Garrod suggests from his observations with patients suffering from certain metabolic diseases that genes dictate the phenotype (= the health condition) of a person 11994400:: the American geneticists G. Beadle and E. Tatum formulate the one gene one enzyme hypothesis based on their experimental results with so-called nutritional mutants of the bread mold Neurospora crassa each of the nutritional mutants lacked one specific enzyme and could only grow on a medium which was supplemented with that nutrient later this hypothesis proofed to be correct and was extended on proteins as well in 1961, the American biochemist M. Nirenberg and co-worker decipher the genetic code which is shared by all forms of life on planet Earth; for this scientific mile stone work he received the Nobel prize in Medicine he chemically synthesized short, artificial RNA molecules with defined sequences (e.g. poly-U or poly-UUC) and used these in a cell-free transcription system consisting of ribosomes and essential factors; ttooddaayy we know that the DNA molecule is organized into special functional units, the so-called genes and each gene on the DNA double helix bears the information for one specific protein or enzyme 1 SAN DIEGO MESA COLLEGE SCHOOL OF NATURAL SCIENCES General Biology Lecture (BIOL107); Instructor: Elmar Schmid, Ph.D. Introduction: the genetic information - laid down in the long sequence of bases, e.g. AGCCGTTAACGT, within the DNA molecule - encodes for a polypeptide chain protein or enzyme can consist of two or more polypeptide chains e.g. the insulin receptor (= a protein which is responsible for the regulation of glucose uptake in our body) is build of two different polypeptide chain which are attached with each other to become a cellular protein or enzyme, this genetic code laid down in the nucleotide sequence of DNA molecules has to be somehow translated by the cell as a pre-requisite of this so-called translation of the genetic information of a gene into a protein, a cell first copies the DNA into a single-stranded polynucleotide molecule, called RNA this special form of DNA replication is called DNA transcription Definition: DNA transcription - transcription is the cellular writing of the genetic information of a gene on the DNA double helix into a piece of RNA molecule let’s look at this first step on the way from genes to cellular proteins in more detail DNA Transcription Transcription = the replication of the genetic information of a gene on the DNA strand into an RNA molecule DNA RNA DNA transcription occurs in the cell nucleus of eukaryotic cells Specific segments of bases along the DNA strand, each with a defined beginning and an end, mark a so-called gene Each gene harbors the genetic information of the DNA strand and codes for a specific protein or enzyme the complete genetic information of an organism is called the genome, which consists of many genes - e.g. the genome of wild mustard (Arabidopsis thaliana) codes for approx. 25,000 genes and contains about 130 million base pairs - e.g. the human genome has an estimated number of 50,000 – 100,000 genes (!) consisting of approx. 3 billion (!!) nucleotides despite this huge number, the human genome has recently been announced to be completely decoded (= sequenced) by a joined effort of American and European research teams! 2 SAN DIEGO MESA COLLEGE SCHOOL OF NATURAL SCIENCES General Biology Lecture (BIOL107); Instructor: Elmar Schmid, Ph.D. TThhee w waayy ffrroom m aa ggeennee ttoo aa cceelllluullaarr pprrootteeiinn during DNA transcription, only one strand of a the double-helix of a gene, the socalled sense-strand, serves as the so-called template for DNA transcription to form the new RNA molecule - certain enzymes unwind and open the DNA double helix and distinct places and create single stranded DNA regions - the distinct places along the DNA strand where transcription begins are called transcription start sites along these unwind and single-stranded DNA regions, new nucleotides are paired according to the Watson-Crick base-pairing rule ( A with T; and G with C) the polymerization of the new nucleotides during the process of DNA transcription is catalyzed by an enzyme called RNA polymerase the RNA polymerase recognizes certain structures on the DNA strand, called enhancers and promoter regions, which tells it where to dock on and where to start the transcription process - In order to begin transcription, RNA polymerase requires a number of so-called transcription factors (TFs), e.g. TFIIA, TFIIB, etc. 3 SAN DIEGO MESA COLLEGE SCHOOL OF NATURAL SCIENCES General Biology Lecture (BIOL107); Instructor: Elmar Schmid, Ph.D. - the RNA polymerase plus the transcription factors recognize and bind to the socalled TATA box these enhancer and promoter regions are always located downstream of the of the so-called transcription start site of a gene - note: the transcription start site of each gene lays in front of a typical ATG triplet nucleotide sequence (= the so-called start codon) which dictates the begin of the later polypeptide chain to be synthesized - the promoter region also dictates which of the two DNA strands is going to be the sense strand after docking to the DNA strand, the RNA polymerase reads only the so-called sense strand of the double-stranded DNA molecule - this is divergent to the earlier introduced features of the DNA- polymerase, which reads both strands of the DNA molecule - an additional divergence to the DNA polymerase is, that the RNA polymerase base-pairs an uracil (U) instead of a thymine (T) with adenine (A) along the DNA strand the resulting RNA molecule is (as the daughter DNA strands after DNA replication) an exact copy of the DNA sense strand sequence The sequence of nucleotides along the RNA molecule dictates the later sequence of amino acids in the polypeptide chain ( see protein translation in the following section) 4 SAN DIEGO MESA COLLEGE SCHOOL OF NATURAL SCIENCES General Biology Lecture (BIOL107); Instructor: Elmar Schmid, Ph.D. The first events of cellular transcription Promoter region ttrraannssccrriippttiioonn ssttaarrtt ssiittee gene docking of first transcription factor (TFII) docking of second transcription factor (TFII) docking of R RN NA A ppoollyym meerraassee + other factors ATP-dependent phosphorylation of R RN NA A ppoollyym meerraassee 5 SAN DIEGO MESA COLLEGE SCHOOL OF NATURAL SCIENCES General Biology Lecture (BIOL107); Instructor: Elmar Schmid, Ph.D. TThhee 33 sstteeppss ooff D DN NA A ttrraannssccrriippttiioonn after activation of the RNA polymerase II by attaching phosphate groups (= phosphorylation), DNA transcription proceeds in three major steps The 3 steps of DNA transcription 11.. IInniittiiaattiioonn An activated RNA polymerase recognizes and binds to a unique DNA sequence located just in front of a gene called the promoter region; It unwinds the helix of the DNA molecule in that region and starts adding new nucleotides along the single-stranded ‘sense- DNA strand’; following the WatsonCrick base-pairing rule The RNA polymerase separates the DNA strands at the appropriate point and bonds the RNA nucleotides as they base-pair along the DNA template. - Like DNA polymerases, RNA polymerases can add nucleotides only to the 3’ end of the growing polymer; - genes are transcribed in 3’->5’ direction, creating a 5’->3’ RNA molecule Bacteria have a single type of RNA polymerase that synthesizes all RNA molecules. In contrast, eukaryotes have three RNA polymerases (I, II, and III) in their nuclei. In Eukaryotes, RNA polymerase II is used for mRNA synthesis. Initiation is a crucial step during DNA transcription since it involves many regulatory proteins, most importantly the highly regulated transcription factors (TFs) (see Figure above & ‘regulation of gene expression’ for more details) 22.. E Elloonnggaattiioonn the RNA synthesis is fully running As RNA polymerase moves along the DNA, it untwists the double helix, 10 to 20 bases at time. The enzyme adds nucleotides to the 3’ end of the growing singlestranded mRNA strand. Behind the point of RNA synthesis, the double helix re-forms and the RNA molecule peels away. the newly formed RNA molecule peels-off the single-stranded DNA template - the two single DNA strands join together again and form the double helix 6 SAN DIEGO MESA COLLEGE SCHOOL OF NATURAL SCIENCES General Biology Lecture (BIOL107); Instructor: Elmar Schmid, Ph.D. 33.. TTeerrm miinnaattiioonn The RNA polymerase proceeds with DNA transcription until it reaches a sequence of bases, the so-called terminator sequence, which consists of a series of stop codons (= UAA, UGA and UAG) In prokaryotes (bacteria), RNA polymerase stops transcription right at the end of the terminator. Both the RNA and DNA is then released. In eukaryotes, the polymerase continues for hundreds of nucleotides past the terminator sequence, AAUAAA. At a point about 10 to 35 nucleotides past this sequence, the pre-mRNA is cut from the enzyme. The RNA polymerase detaches from the newly formed RNA molecule and the corresponding gene 7 SAN DIEGO MESA COLLEGE SCHOOL OF NATURAL SCIENCES General Biology Lecture (BIOL107); Instructor: Elmar Schmid, Ph.D. The three steps of DNA transcription 3 different kinds of RNA molecules are assembled (= polymerized) by this transcription mechanism 1. messenger RNA (= mRNA) 2. transfer RNA (= tRNA) 3. ribosomal RNA (= rRNA) 8 SAN DIEGO MESA COLLEGE SCHOOL OF NATURAL SCIENCES General Biology Lecture (BIOL107); Instructor: Elmar Schmid, Ph.D. all three play a crucial role in the final cellular scenario on the way from the gene to a functional protein or enzyme 1. TThhee m meesssseennggeerr R RN NA A ((== m mR RN NA A)) mRNA is a single-stranded polynucleotide strand, which delivers the encoded amino acid sequence of the future protein or enzyme out of the nucleus to the ribosomes in eukaryotic cells, the mRNA leaves the cell nucleus via the nuclear openings or pores in eeuukkaarryyoottiicc cceellllss, however, only a shortened version or a so-called spliced form of the original RNA strand (= nRNA or pre-mRNA) reaches the ribosome as mRNA during RNA splicing, the so-called Intron sequences are cleaved or cut out of the primary DNA transcription product (= pre-mRNA); only the assembled exon sequences are send to the ribosomes in the cell nucleus of eukaryotic cells, only about 1% (!!) of the DNA actually encodes for a certain protein or enzyme product; these coding sequence of cellular DNA are called exons, they are interrupted by long stretches of noncoding DNA, the so-called introns during DNA transcription into RNA, the cell first transcribes all of the DNA, including the exons and introns of the gene, into a complementary RNA copy; this primary transcription product is called ‘nuclear RNA’ (= nRNA) or pre-promessenger-RNA the cell then removes in a second step all information-less (= non-coding) introns from the mRNA by a process called RNA splicing; this edited ( or spliced RNA) sequence is called pro-mRNA the spliced mRNA (or so-called pro-mRNA) molecule is further processed and trimmed by the cell to prevent rapid degradation of the molecule by cellular enzymes called RNAses At the 5’ end of the pre-mRNA molecule, a modified form of guanine is added, to form the socalled 5’ cap. This chemical modification helps to protect mRNA from hydrolytic enzymes. It also functions as an “attach here” signal for ribosomes. Other enzymes attach consecutive adenine nucleotides to the 3’-end of the promRNA molecule to form a so-called poly-A sequence or poly-A tail in a process called poly-adenylation - both modified mRNA end modifications help to prevent the otherwise very rapid degradation of this very ‘fragile’ cellular molecule 9 SAN DIEGO MESA COLLEGE SCHOOL OF NATURAL SCIENCES General Biology Lecture (BIOL107); Instructor: Elmar Schmid, Ph.D. The thus spliced and processed final version of the mRNA travels from the nucleus into the cytosol to the ribosomes; the place of cellular protein synthesis, called ribosomes - in eukaryotic cells the ribosomes are located on the surface of the rough endoplasmic reticulum (rER) - in bacteria they swim freely in the cytosol The Intron-Exon structure of eukaryotic DNA 10 SAN DIEGO MESA COLLEGE SCHOOL OF NATURAL SCIENCES General Biology Lecture (BIOL107); Instructor: Elmar Schmid, Ph.D. 11 SAN DIEGO MESA COLLEGE SCHOOL OF NATURAL SCIENCES General Biology Lecture (BIOL107); Instructor: Elmar Schmid, Ph.D. Splicing of eukaryotic, but not prokaryotic RNA S Scchheem mee:: E Euukkaarryyoottiicc R RN NA AS Spplliicciinngg 12 SAN DIEGO MESA COLLEGE SCHOOL OF NATURAL SCIENCES General Biology Lecture (BIOL107); Instructor: Elmar Schmid, Ph.D. 2. The transfer-RNA (= tRNA) the tRNA molecule is a short polynucleotide chain consisting of only about 80 nucleotides the tRNA molecules function as the cellular interpreter and decoder molecules which translate the genetic language (= genetic code) into the amino acid language (= sequence) ‘t’ stands for transfer, because tRNAs transport and transfer the cellular amino acids to the mRNA strand, which has docked at the ribosomes at the ribosomes they convert the 3 letter codes (= codon) of the nucleotides on the mRNA molecule into the one-letter amino acid words a tRNA molecule picks up “its amino acid’ and transfers it to its corresponding codon on the mRNA strand (following the genetic code) tRNAs have a complex 3-dimensional structure which has two major so-called interaction domains (see Figure below) 11.. an amino acid attachment site the site on the tRNA molecule where the corresponding amino acid is covalently attached to the tRNA 2. a so-called anti-codon site which is complementary to a codon triplet on the mRNA strand with which it base-pairs during the translation process the transfer of the matching amino acid onto the corresponding tRNA is preformed by a specialized enzyme under consumption of ATP - this tRNA-amino acid complex then delivers its load to the growing polypeptide chain at the ribosome 13 SAN DIEGO MESA COLLEGE SCHOOL OF NATURAL SCIENCES General Biology Lecture (BIOL107); Instructor: Elmar Schmid, Ph.D. Functional domains (left figure) & 3-dimensional structure (right figure) of a tRNA molecule aam miinnoo aacciidd-attachment site m mR RN NA A _______ GCU_____________ (codon) aannttii--ccooddoonn site 3. The ribosomal RNA molecule (= rRNA) it is the RNA molecules which are crucial structural and functional part of the ribosomes; it actively takes part in the many catalytic activities of this proteinsynthesizing protein complex - two forms of rRNA with different length exist in prokaryotic organisms (= 16SrRNA & 23S-rRNA) and in eukaryotic organisms (= 18S-rRNA & 28S-rRNA) - they are synthesized (= transcribed) in high amounts in a special region of the nucleus, called the nucleolus 14 SAN DIEGO MESA COLLEGE SCHOOL OF NATURAL SCIENCES General Biology Lecture (BIOL107); Instructor: Elmar Schmid, Ph.D. Micro-RNA - - in the past years, scientist discovered additional forms of RNA molecules in cells of fungi, plants and animals, the small, so-called micro-RNAs, which mostly unknown biological function the so-called silencer RNA or (siRNA) molecules seem to have an important function in the shutting-off (= silencing) of gene expression and to prevent viral replication in cells The genetic code of life not like in computers, where technical engineers use a 2 numbered, so-called binary code, to encrypt and save technical data and information, all biological organisms use a sequence of 3 nucleotides on the DNA/RNA molecule - or a socalled triplet - code to encode the information of life - each triplet code stands for an individual amino acid - the 3 nucleotide unit is also called a codon e.g. GCT A G G GTC TTrriipplleett ccooddoonn aam miinnoo aacciidd 11 aam miinnoo aacciidd 22 aam miinnoo aacciidd 33 . . . etc. the whole genetic information of an organism is written down as (triplet) codons, which are translated (= translation!) into an amino acid sequence - the sequence of triplet codons is the “chemical language” of DNA and RNA triplets (not duplets) of bases provide the smallest unit of information which is able to specify all the known 20 amino acids - duplets could only code for 42 = 16 different amino acids, while a triplet code enables 43 = 64 different code words for amino acids! since the triplet code gives the cell more coding possibilities than it actually needs to code, there are multiple triplets coding for the same of the existing 20 amino acids; scientists say the genetic code is redundant the different triplets coding for a specific amino acid are written down in the socalled genetic code of the DNA molecule Milestone experiments of science The genetic code was deciphered by the elegant and milestone experiments conducted by the American biochemist M. Nirenberg in 1961 - he chemically synthesized short, artificial RNA molecules with defined sequences (e.g. poly-U or poly-UUC) and used these in a (by that time established) cell-free transcription system - a cell-free transcription system consists of ribosomes and essential biomolecules ( co-factors) 15 SAN DIEGO MESA COLLEGE SCHOOL OF NATURAL SCIENCES General Biology Lecture (BIOL107); Instructor: Elmar Schmid, Ph.D. by varying the RNA sequences and determining the incorporated amino acids of the resulting polypeptide chain, Nirenberg retrieved the 61 of the 64 theoretically possible triplet codes for each amino acid; he unraveled the genetic code (see Chart below) he revealed that the codon A AU UG G has dual function: 1. 2. it codes for the amino acid methionine it is the start signal or ssttaarrtt codon for the transcription machinery today we know, that ALL polypeptides manufactured in living organisms, start with the amino acid methionine! UA the three other codons (U AA A,, U UA AG G and U UG GA A) are not coding for an amino acid; they set the stop signal for the transcription enzymes; they are also called ssttoopp codons almost all of the genetic code is shared by all organisms on Earth; from the simplest bacteria to the most complex plants and animals; different forms of life use the same genetic code to translate their DNA sequence into a protein - that means that bacteria or yeast can translate human genetic messages into functional proteins - this is of great importance for modern Biotechnology, where bacteria or yeast cells are used to produce large amounts of proteins or peptides for e.g. bioanalytical tests or treatment of human disease, e.g.: Erythropoietin (EPO) Interferons Insulin anemia certain cancers diabetes The genetic code & Evolution “The genetic code is not frozen in time but obviously evolves …” in the recent years, however, scientists identified at least 16 variants to the code while in most biological organisms the codon CUG is translated into the amino acid leucine (Leu), several biological species have been discovered where this codon translates into a different amino acid - e.g. in the fungal Candida species, CUG translates into serine (Ser) - e.g. the green algae Acetabularia translates the stop codons UAG & UAA into glycine (Gly) - e.g. in some organisms the standard stop codon UGA codes for the rare amino acid selenocysteine - e.g. mitochondria and many bacterial species have large variations to the “classical genetic code” unraveled by Nirenberg in the yeast mitochondrial code, 4 of 6 codons that normally encode leucine (Leu) code for threonine (Thr) 16 SAN DIEGO MESA COLLEGE SCHOOL OF NATURAL SCIENCES General Biology Lecture (BIOL107); Instructor: Elmar Schmid, Ph.D. today it becomes more and more obvious, that the genetic code is not a “static product of chance” with randomly assigned codons for each of the 20 amino acids, but rather the product of a molecular evolutionary process the assigned codons are arranged well in terms of ensuring that errors occurring during protein translation do not lead to catastrophic consequences, means the synthesis of dysfunctional proteins The genetic code chart with the knowledge of the complete DNA sequences for many organisms, including humans, we are now able to read the genetic information and master plans for all life components of these species 17 SAN DIEGO MESA COLLEGE SCHOOL OF NATURAL SCIENCES General Biology Lecture (BIOL107); Instructor: Elmar Schmid, Ph.D. P Prrootteeiinn ttrraannssllaattiioonn –– TThhee eevveennttss aatt tthhee rriibboossoom mee the last step of the conversion of the genetic information (stored in a linear nucleotide sequence) on the DNA into a functional polypeptide chain (called protein or enzyme) biologists call protein translation the process of protein translation occurs at special protein structures in the cell, called ribosomes ribosomes are the cell’s polypeptide manufacturing belt in comparison to cell organelles, ribosomes are small, but not less complex structures, consisting of two unequally sized sub-units, referred to as large and small sub-units, which fit closely together as seen in the Figures below a sub-unit is composed of a complex between RNA molecules and proteins; each sub-unit contains at least one ribosomal RNA (rRNA) sub-unit and a large quantity of ribosomal proteins ribosomes in bacteria are located in the cytosol, while in eukaryotic cells they are found on the surface of a special organelle, the rough endoplasmic reticulum (rER) The location of ribosomes in a eukaryotic cell 18 SAN DIEGO MESA COLLEGE SCHOOL OF NATURAL SCIENCES General Biology Lecture (BIOL107); Instructor: Elmar Schmid, Ph.D. ribosomes consist of 2 major components: 1. Protein the protein part consists of two globular protein sub-units, a so-called small and large sub-unit the protein sub-units have a different size in bacteria and eukaryotic cells (see Figures below) 2. rRNA three rRNA molecules (5S, 16S & 23S-rRNA) ( in prokaryotes) or four rRNA molecules (7S, 18S & 28S-rRNA) ( in eukaryotes), each with a different are attached with certain regions of both protein sub-units more than half of the weight of a ribosome is RNA, and there is clear evidence that the ribosomal RNA (rRNA) molecules have catalytic activity; rRNA plays a central part in the attachment of amino acids and the extension of the polypeptide chain many more proteins are known which interact or bind to the ribosome The ribosome structures the Figures show only the protein sub-units of a eukaryotic (top) and a bacterial (bottom) ribosome 19 SAN DIEGO MESA COLLEGE SCHOOL OF NATURAL SCIENCES General Biology Lecture (BIOL107); Instructor: Elmar Schmid, Ph.D. during the cellular process of translation, several components or molecules dock at these two ribosomal structures a ribosome contains three binding sites for RNA molecules: - one for mRNA and three for tRNAs - one site, called the peptidyl-tRNA-binding site, or P-site, holds the tRNA molecule that is linked to the growing end of the polypeptide chain - another site, called the aminoacyl-tRNA-binding site, or A-site, holds the incoming tRNA molecule charged with a covalently bound amino acid (see Figure below) - the empty (E) site holds the discharged tRNA which gave off its bound amino acid during the transpeptidylation process I. - 2 amino acid-loaded tRNA (= aminoacyl tRNA) molecules they bind to the so-called P and A sites of the small subunit the P site holds the growing polypeptide chain the A site is the docking site for the new and matching tRNA molecule with its attached amino acid (= aminoacyl-tRNA) bacterial ribosome during protein synthesis II. one mRNA strand - binds also to the small sub-unit of the ribosome - its correct alignment with the A- and P-sites is enabled with the help of the ribosomal RNA (= rRNA) - the 2 anti-codon sequences of two closely neighbored and ‘loaded’ tRNA molecules base-pair with the corresponding codon on the bound mRNA strand - all three biomolecules are closely brought and hold together in a cavity build by the two ribosomal sub-units 20 SAN DIEGO MESA COLLEGE SCHOOL OF NATURAL SCIENCES General Biology Lecture (BIOL107); Instructor: Elmar Schmid, Ph.D. - the ribosome assures that the 2 anti-codon sequences of two closely neighbored and amino acid-‘loaded’ tRNA molecules base-pair with the corresponding codon on the bound mRNA strand all three biomolecules are closely brought and hold together in a cavity build by the two ribosomal subunits at the begin of the translation process, the mRNA binds to the small ribosomal sub-unit via a so-called recognition sequence located at the begin and end of the RNA strand The tRNA- & mRNA-binding sites on the ribosome & RNA alignment 21 SAN DIEGO MESA COLLEGE SCHOOL OF NATURAL SCIENCES General Biology Lecture (BIOL107); Instructor: Elmar Schmid, Ph.D. 3 -dimensional prokaryotic ribosome structure computer-assisted image based on X-ray crystallographic data with a purified ribosome Large ribosomal sub-unit Small ribosomal sub-unit tRNA (waiting) tRNA (A-site) tRNA (P-site) 22 SAN DIEGO MESA COLLEGE SCHOOL OF NATURAL SCIENCES General Biology Lecture (BIOL107); Instructor: Elmar Schmid, Ph.D. The 3 major steps of translation of mRNA at the ribosome generally, translation proceeds in a highly ordered process. First all components necessary for translation are brought together in a process called Initiation. Secondly, in a process called Elongation, the polypeptide chain grows in a distinct direction and becomes longer. Finally, the chain of amino acids gets released from the ribosome in a process called Termination 1. IInniittiiaattiioonn or codon recognition - a so-called pre-initiation ribosome complex forms; it consists of: 1. 2. 3. 4. - the small ribosome sub-unit (= 40S) the mRNA a GTP molecule an initiation protein/factor (= eIF-2) the large ribosomal sub-unit (= 60S) associates with this pre- initiation complex to form the 80S complex this step requires another protein factor, called eIF-5 in Prokaryotes, the 16S-rRNA component of the ribosome recognizes the socalled Shine-Dalgarno sequence on the “captured” mRNA strand the anti-codon of a “loaded” or so-called amino-acyl tRNA molecule basepairs with its ‘correct’ mRNA codon in the A-site of the ribosome this first step requires in both, Prokaryotes and Eukaryotes, a specific initiator-tRNA, called tRNAmetI (in prokaryotes the methionine of the initiator-tRNA is formylated) - the initiator tRNA molecule base-pairs with the AUG start codon of the mRNA strand in the P-site of the ribosome; it incorporates the initial amino acid methionine into the new protein - Initiation requires so-called Initiation factors (IF); IF’s are proteins which enable the correct and accurate positioning of certain players at the ribosome e.g. eIF-1, eIF-2, eIF-4E (or GEF), eIF-4E, eIF-4A 23 SAN DIEGO MESA COLLEGE SCHOOL OF NATURAL SCIENCES General Biology Lecture (BIOL107); Instructor: Elmar Schmid, Ph.D. Translation Initiation 22.. E Elloonnggaattiioonn or peptide bond formation - - - - during elongation the new polypeptide strand forms and becomes longer (= elongates) Elongation at the ribosome occurs in a cyclical manner at the end of one complete round of amino acid addition, the A-site of the ribosome will be empty again and the ribosome ready for the docking of the new, matching amino acyl-tRNA molecule the matching aminoacyl-tRNA (= the aminoacyl-tRNA with the matching anticodon site) is brought to the empty A-site of the ribosome by an elongation factor (EF) complex, called eEF-1-GTP the amino acid/peptide attached with the tRNA in the P-site is transferred to the amino group of the aminoacyl-tRNA located in the A-site; this step of translation, which is also called transpeptidylation, is catalyzed by an enzyme, called peptidyl transferase at least parts of this chemical reaction seem to be catalyzed by the enzyme-activity of the 23S-rRNA !! (RNA can work as an enzyme = ribozyme activity !!) the polypeptide strand separates from the tRNA molecule in the P-site and attaches to the amino acid bound on the other tRNA in the A-site a covalent peptide bond is formed between the two neighboring amino acids 24 SAN DIEGO MESA COLLEGE SCHOOL OF NATURAL SCIENCES General Biology Lecture (BIOL107); Instructor: Elmar Schmid, Ph.D. Translocation the now ‘empty’ tRNA in the P-site leaves the ribosome and the ribosome moves the tRNA (including the attached polypeptide chain) in the A-site over to the vacant P-site this step is again catalyzed by an enzyme, called eEF-2 and requires energy in form of the ATP-like molecule GTP during this translocation the anti-codon of the remaining tRNA remains paired with its codon on the mRNA since the mRNA also moves over to the P-site it exposes the next codon to the anti-codon of the new incoming and ‘loaded’ tRNA the process of polypeptide elongation starts again with step 1 until a stop codon on the mRNA strand arrives at the ribosomal A-site following this translation scheme, the whole mRNA molecule is translated in 5’ 3’ direction! 25 SAN DIEGO MESA COLLEGE SCHOOL OF NATURAL SCIENCES General Biology Lecture (BIOL107); Instructor: Elmar Schmid, Ph.D. The elongation cycle of cellular translation 26 SAN DIEGO MESA COLLEGE SCHOOL OF NATURAL SCIENCES General Biology Lecture (BIOL107); Instructor: Elmar Schmid, Ph.D. 27 SAN DIEGO MESA COLLEGE SCHOOL OF NATURAL SCIENCES General Biology Lecture (BIOL107); Instructor: Elmar Schmid, Ph.D. The three steps of elongation 33.. TTeerrm miinnaattiioonn - - - the signals for ending translation at the ribosome are the same for Prokaryotes and Eukaryotes in both cases, the termination signal is set by the appearance of a combination of the so-called stop codons at the 3’-end of the RNA molecule; the stop codons are UAG, UUA and UGA the termination codons are recognized by certain so-called releasing factors (= eRFs) the releasing factors (eRF1 or eRF-2) bind to the empty A-site on the ribosome; as a consequence the peptidyltransferase moves the peptide chain (bound in the P-site) to water instead of an aminoacyl-tRNA! the complete polypeptide chain (approx. 100 amino acids long!) leaves the ribosome which is freed from the last tRNA molecule in the P-site 28 SAN DIEGO MESA COLLEGE SCHOOL OF NATURAL SCIENCES General Biology Lecture (BIOL107); Instructor: Elmar Schmid, Ph.D. - the inactive ribosome releases its bound mRNA and separates into its two sub-units again Termination… the final scenario of protein translation Since the ribosomes of eukaryotic cells are located on the membrane of the rER, the newly formed polypeptide chain doesn’t leave into the cytosol but is tunneled through the phospholipid membrane of the rER into the interior space (= lumen) of the rER - in the rER lumen, the freshly synthesized polypeptide chain is properly folded into its final functional 3-dimensional shape with the help of special folding helper enzymes, called chaperones - most of the polypeptides or proteins are even further trimmed (= processed) within the rER lumen in a cellular process called post-translational modification e.g. in many cases sugar molecules are attached to the proteins which is called glycosylation; protein glycosylation plays a fundamental role in the friend or foe-recognition process of our immune system or in the formation of the human ABO blood groups e.g. many so-called signaling proteins become modified by attachment of certain fatty acids in a process called farnesylation or palmitoylation e.g. some polypeptides, such as insulin, are processed by chopping parts of the chain off before they are released into the cell or blood stream the ‘cellular end control’ for newly synthesized proteins is also found in the interior space of the rER; - defect or not properly folded proteins are recognized and sorted-out by ‘cellular watchdogs’ called heat shock protein (hsp); 29 SAN DIEGO MESA COLLEGE SCHOOL OF NATURAL SCIENCES General Biology Lecture (BIOL107); Instructor: Elmar Schmid, Ph.D. they are then send to another organelle, called proteasome, where they are degraded and recycled Usually mRNA molecules are translated simultaneously by a number of ribosomes on the rER Ribosomal translation is a tremendously fast cellular process it takes a cell less than a minute to synthesize an average-sized polypeptide of about 100 amino acids! DNA transcription and translation is usually not permanently happening in a cell and does not involve all cellular genes at the same time Both processes are rather highly regulated cellular processes which are controlled at different levels Protein translation & Mistakes or Errors As seemingly perfect the cellular “protein translation machinery” may appear to the human observer, it is not free of eventual flaws and errors occurring during the intricate protein translation process at the ribosome Mistakes in translation of the genetic information of the mRNA molecule into a corresponding amino acid sequence occurs most frequently at the third codon position The third codon position (e.g. the G in CUG) is the most error prone spot of the codon since it has the weakest binding affinity for the corresponding tRNA molecule “The third codons form the weakest link of the genetic code…” Scientists say, that the genetic code “wobbles” in the third codon position Since, interestingly, codons sharing 2 out of 3 bases tend to code for amino acids with the same physico-chemical properties (e.g. hydrophobicity, polarity, net charges), the consequences of a “codon wobbling event” are usually minor for the resulting amino acid chain A point mutation of DNA that results in replacement of a pair of complimentary nucleotides with another nucleotide pair is called a base-pair substitution. Some base-pair substitutions have little or no impact on protein function. 30 SAN DIEGO MESA COLLEGE SCHOOL OF NATURAL SCIENCES General Biology Lecture (BIOL107); Instructor: Elmar Schmid, Ph.D. In silent mutations, alterations of nucleotides still indicate the same amino acids because of redundancy in the genetic code. - Other changes lead to switches from one amino acid to another with similar properties - Still other mutations may occur in a region where the exact amino acid sequence is not essential for function Other base-pair substitutions cause a readily detectable change in a protein. These are usually detrimental but can occasionally lead to an improved protein or one with novel capabilities. Changes in amino acids at crucial sites, especially active sites, are likely to impact function. 31 SAN DIEGO MESA COLLEGE SCHOOL OF NATURAL SCIENCES General Biology Lecture (BIOL107); Instructor: Elmar Schmid, Ph.D. Missense mutations are those that still code for an amino acid but change the indicated amino acid. Nonsense mutations change an amino acid codon into a stop codon, nearly always leading to a nonfunctional protein. 32 SAN DIEGO MESA COLLEGE SCHOOL OF NATURAL SCIENCES General Biology Lecture (BIOL107); Instructor: Elmar Schmid, Ph.D. Insertions and deletions are additions or losses of nucleotide pairs in a gene. These have a disastrous effect on the resulting protein more often than substitutions do. - many inherited human disorders, such as Cri-du-Chat syndrome are caused by this type of mutation Unless these mutations occur in multiples of three, they cause a frameshift mutation. All the nucleotides downstream of the deletion or insertion will be improperly grouped into codons. The result will be extensive missense, ending sooner or later in nonsense - premature termination. However, the surprising non-random codon arrangement of the genetic code (see sections above) assures that the consequences of a wobbling event in the third codon site during protein translation does NOT lead to an incorporation of an amino acid with completely different physico-chemical properties incase of a mutation Based on this interesting observation, whereas the genetic code of life bears a non-random assignment of codons with their corresponding amino acids, scientists hypothesize today, that the genetic code is the product of natural selection on the molecular level The genetic code is dynamic and evolved through natural selection Of all possible random assignments of 64 codons with the existing 20 amino acids, the genetic code unraveled by M. Nirenberg, turned out to be the very code used by almost all biological organisms on planet Earth to assure the least risk of producing aberrant and dysfunctional proteins during protein translation due to intrinsic “wobbling”, mutations or viruses Today, we have to assume that not only the intricate shapes, morphologies and traits of biological organisms evolved over long periods of time, but that even the genetic code has to be seen as the product of evolution The genetic code evolved by natural selection as the most favorable of all possible genetic codes to life and survived the billions of years since the rise of the first life forms on planet Earth Further Readings: 1. H. Lodish, A. Berk, S.L. Zipursky, P. Matsudaira, D. Baltimore & J. Darnell. “Molecular Cell Biology” (4th edition: W.H. Freeman): Chapter 4. 2. S.J. Freeland & L.D. Hurst “Evolution Encoded” Scientific American (April 2004): 84-91 3. S.J. Freeland & L.D. Hurst. “The genetic code in one in a million.” J. Molecular Evolution 47(3): 238-248 (1998) 33 SAN DIEGO MESA COLLEGE SCHOOL OF NATURAL SCIENCES General Biology Lecture (BIOL107); Instructor: Elmar Schmid, Ph.D. Inhibition of protein translation by antibiotics and toxins Since DNA transcription and protein translation are enormously complex cellular process, involving many “molecular players”, it is not too surprising that both processes are enormously vulnerable to many natural or synthetic compounds many of these transcription or translation blockers (or inhibitors) we humans know and fear (some since ancient times) as famous or infamous toxins or poisons many of our modern antibiotics, we use for treatment of bacterial or fungal infections, function through blockade (= inhibition) of cellular translation different antibiotics interfere and block at different levels of translation ( see Table below) Known Inhibitors of Cellular Protein Translation Antibiotic or Toxin C Chhlloorraam mpphheenniiccooll S Sttrreeppttoom myycciinn TTeettrraaccyycclliinnee E Erryytthhrroom myycciinn Target of protein translation inhibits the bacterial peptidyl transferase inhibits the prokaryotic peptide chain initiation inhibits the binding of the aminoacyl-tRNA to the ribosome small unit in prokaryotes inhibits the translocation of the mRNA through the ribosome large sub-unit in prokaryotes resembles in shape an aminoacyltRNA molecule and interferes with peptide transfer; leads to premature termination at the ribosome attaches an ADP-ribose molecule to the eEF-2 in eukaryotic cells and inactivates this elongation factor this plant-derived toxin catalyzes the cleavage of the eukaryotic large subunit rRNA from the ribosome inhibits the eukaryotic Peptidyltransferase P Puurroom myycciinn D Diipphhtteerriiaa ttooxxiinn R Riicciinn C Cyycclloohheexxiim miiddee 34