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Lecture PowerPoint to accompany Molecular Biology Fourth Edition Robert F. Weaver Chapter 18 The Mechanism of Translation II: Elongation and Termination Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 18.1 Direction of Polypeptide Synthesis and mRNA Translation • Messenger RNAs are read in the 5’3’ direction • This is the same direction in which they are synthesized • Proteins are made in the aminocarboxyl direction • This means that the amino terminal amino acid is added first 18-2 Strategy to Determine Direction of Translation 18-3 18.2 The Genetic Code • The term genetic code refers to the set of 3-base code words (codons) in mRNA that represent the 20 amino acids in proteins • Basic questions were answered about translation in the process of “breaking” the genetic code 18-4 Nonoverlapping Codons • Each base is part of at most one codon in nonoverlapping codons • In an overlapping code, one base may be part of two or even three codones 18-5 No Gaps in the Code • If the code contained untranslated gaps or “commas”, mutations adding or subtracting a base from the message might change a few codons • Would still expect ribosome to be back “on track” after the next such comma • Mutations might frequently be lethal – Many cases of mutations should occur just before a comma and have little, if any, effect 18-6 Frameshift Mutations Frameshift mutations • Translation starts • Insert an extra base AUGCAGCCAACG AUXGCAGCCAACG – Extra base changes not only the codon in which is appears, but every codon from that point on – The reading frame has shifted one base to the left Code with commas • Each codon is flanked by one or more untranslated bases – Commas would serve to set off each codon so that ribosomes recognize it • Translation starts • Insert an extra base AUGZCAGZCCAZACGZ AUXGZCAGZCCAZACGZ – First codon wrong, all others separated by Z, translated 18-7 normally Frameshift Mutation Sequences 18-8 The Triplet Code • The genetic code is a set of three-base code words, or codons – In mRNA, codons instruct the ribosome to incorporate specific amino acids into a polypeptide • Code is nonoverlapping – Each base is part of only one codon • Devoid of gaps or commas – Each base in the coding region of an mRNA is part of a codon 18-9 Coding Properties of Synthetic mRNAs 18-10 Breaking the Code • The genetic code was broken – Using: • Synthetic messengers • Synthetic trinucleotides – Then observing: • Polypeptides synthesized • Aminoacyl-tRNAs bound to ribosomes • There are 64 codons – 3 are stop signals – Remainder code for amino acids – The genetic code is highly degenerate 18-11 The Genetic Code 18-12 Unusual Base Pairs Between Codon and Anticodon Degeneracy of genetic code is accommodated by: – Isoaccepting species of tRNA: bind same amino acid, but recognize different codons – Wobble, the 3rd base of a codon is allowed to move slightly from its normal position to form a non-Watson-Crick base pair with the anticodon – Wobble allows same aminoacyl-tRNA to pair with more than one codon 18-13 Wobble Base Pairs • Compare standard Watson-Crick base pairing with wobble base pairs • Wobble pairs are: – G-U – I-A 18-14 Wobble Position 18-15 Almost Universal Code • Genetic code is NOT strictly universal • Certain eukaryotic nuclei and mitochondria along with at least one bacterium – Codons cause termination in standard genetic code can code for amino acids Trp, Glu – Mitochondrial genomes and nuclei of at least one yeast have sense of codon changed from one amino acid to another • Deviant codes are still closely related to standard one from which they evolved • Genetic code a frozen accident or the product of evolution – Ability to cope with mutations evolution 18-16 Deviations from “Universal” Genetic Code 18-17 18.3 The Elongation Mechanism Elongation takes place in three steps: 1. EF-Tu with GTP binds aminoacyl-tRNA to the ribosomal A site 2. Peptidyl transferase forms a peptide bond between peptide in P site and newly arrived aminoacyl-tRNA in the A site Lengthens peptide by one amino acid and shifts it to the A site 3. EF-G with GTP translocates the growing peptidyl-tRNA with its mRNA codon to the P site 18-18 Elongation in Translation 18-19 A Three-Site Model of the Ribosome • Puromycin – – – – Resembles an aminoacyl-tRNA Can bind to the A site Couple with the peptide in the P site Release it as peptidyl puromycin • If peptidyl-tRNA is in the A site, puromycin will not bind to ribosome, peptide will not be released • Two sites are defined on the ribosome: – Puromycin-reactive site (P) – Puromycin unreactive site (A) • 3rd site (E) for deacylated tRNA bind to E site as exits ribosome 18-20 Puromycin Structure and Activity 18-21 Protein Factors and Peptide Bond Formation • One factor is T, transfer – It transfers aminoacyl-tRNAs to the ribosome – Actually 2 different proteins • Tu, u stands for unstable • Ts, s stands for stable • Second factor is G, GTPase activity • Factors EF-Tu and EF-Ts are involved in the first elongation step • Factor EF-g participates in the third step 18-22 Elongation Step 1 Binding aminoacyl-tRNA to A site of ribosome • Ternary complex formed from: – – – EF-Tu Aminoacyl-tRNA GTP • Delivers aminoacyl-tRNA to ribosome A site without hydrolysis of GTP • Next step: – – – EF-Tu hydrolyzes GTP Ribosome-dependent GTPase activity EF-Tu-GDP complex dissociates from ribosome • Addition of aminoacyl-tRNA reconstitutes ternary complex for another round of translation elongation 18-23 Aminoacyl-tRNA Binding to Ribosome A Site 18-24 Proofreading • Protein synthesis accuracy comes from charging tRNAs with correct amino acids • Proofreading is correcting translation by rejecting an incorrect aminoacyl-tRNA before it can donate its amino acid • Protein-synthesizing machinery achieves accuracy during elongation in two steps 18-25 Protein-Synthesizing Machinery • Two steps achieve accuracy: – Gets rid of ternary complexes bearing wrong aminoacyl-tRNA before GTP hydrolysis – If this screen fails, still eliminate incorrect aminoacyltRNA in the proofreading step before wrong amino acid is incorporated into growing protein chain • Steps rely on weakness of incorrect codonanticodon base pairing to ensure dissociation occurs more rapidly than either GTP hydrolysis or peptide bond formation 18-26 Proofreading Balance • Balance between speed and accuracy of translation is delicate – If peptide bond formation goes too fast • Incorrect aminoacyl-tRNAs do not have enough time to leave the ribosome • Incorrect amino acids are incorporated into proteins – If translation goes too slowly • Proteins are not made fast enough for the organism to grow successfully • Actual error rate, ~0.01% per amino acid is a good balance between speed and accuracy 18-27 Elongation Step 2 • One the initiation factors and EF-Tu have done their jobs, the ribosome has fMettRNA in the P site and aminoacyl-tRNA in the A site • Now form the first peptide bond • No new elongation factors participate in this event • Ribosome contains the enzymatic activity, peptidyl transferase, that forms peptide bond 18-28 Assay for Peptidyl Transferase 18-29 Peptide Bond Formation • The peptidyl transferase resides on the 50S ribosomal particle • Minimum components necessary for activity are 23S rRNA and proteins L2 and L3 • 23S rRNA is at the catalytic center of peptidyl transferase 18-30 Elongation Step 3 • When peptidyl transferase has worked: – Ribosome has peptidyl-tRNA in the A site – Deacylated tRNA in the P site • Translocation, next step, moves mRNA and peptidyl-tRNA one codon’s length through the ribosome – Places peptidyl-tRNA in the P site – Ejects the deacylated tRNA – Process requires elongation factor EF-G which hydrolyzes GTP after translocation is complete 18-31 Three-Nucleotide Movement Each translocation event moves the mRNA on codon length, or 3 nt through the ribosome 18-32 Role of GTP and EF-G • GTP and EF-G are necessary for translocation – Translocation activity appears to be inherent in the ribosome – This activity can be expressed without EF-G and GTP • GTP hydrolysis – Precedes translocation – Significantly accelerate translocation • New round of elongation occurs if: – EF-G must be released from the ribosome – Release depends on GTP hydrolysis 18-33 GTPases and Translation • Some translation factors harness GTP energy to catalyze molecular motions • These factors belong to a large class of G proteins – Activated by GTP – Have intrinsic GTPase activity activated by an external factor (GAP) – Inactivated when they cleave their own GTP to GDP – Reactivated by another external factor (guanine nucleotide exchange protein) that replaces GDP with GTP 18-34 G Protein Features • Bind GTP and GDP • Cycle among 3 conformational states – Depends on whether bound to: • GDP • GTP • Neither – Conformational state determine activity • Activated to carry out functionality when bound to GTP • Intrinsic GTPase activity 18-35 More G Protein Features • GTPase activity stimulated by GTPase activator protein (GAP) – When GAP stimulates GTPase cleave GTP to GDP – Results in self inactivation • Reactivation by guanine nucleotide exchange protein – Removes GDP from inactive G protein – Allows another molecule of GTP to bind – Example of guanine nucleotide exchange protein is EF-Ts 18-36 Structures of EF-Tu and EF-G • Three-dimensional shapes determined by x-ray crystallography: – EF-Tu-tRNA-GDPNP ternary complex – EF-G-GDP binary complex • As predicted, the shapes are very similar 18-37 18.4 Termination • Elongation cycle repeats over and over – Adds amino acids one at a time – Grows the polypeptide product • Finally ribosome encounters a stop codon – Stop codon signals time for last step – Translation last step is termination 18-38 Termination Codons • Three codons are the natural stop signals at the ends of coding regions in mRNA – UAG – UAA – UGA • Mutations can create termination codons within an mRNA causing premature termination of translation – Amber mutation creates UAG – Ochre mutation creates UAA – Opal mutation creates UGA 18-39 Amber Mutation Effects in a Fused Gene 18-40 Termination Mutations • Amber mutations are caused by mutagens that give rise to missense mutations • Ochre and opal mutations do not respond to the same suppressors as do the amber mutations – Ochre mutations have their own suppressors – Opal mutations also have unique suppressors 18-41 Termination Mutations 18-42 Stop Codon Suppression • Most suppressor tRNAs have altered anticodons: – Recognize stop codons – Prevent termination by inserting an amino acid – Allow ribosome to move on to the next codon 18-43 Release Factors • Prokaryotic translation termination is mediated by 3 factors: – RF1 recognizes UAA and UAG – RF2 recognizes UAA and UGA – RF3 is a GTP-binding protein facilitating binding of RF1 and RF2 to the ribosome • Eukaryotes has 2 release factors: – eRF1 recognizes all 3 termination codons – eRF3 is a ribosome-dependent GTPase helping eRF1 release the finished polypeptide 18-44 Release Factor Assays 18-45 Dealing with Aberrant Termination • Two kinds of aberrant mRNAs can lead to aberrant termination – Nonsense mutations can occur that cause premature termination – Some mRNAs (non-stop mRNAs) lack termination codons • Synthesis of mRNA was aborted upstream of termination codon • Ribosomes translate through non-stop mRNAs and then stall • Both events cause problems in the cell yielding incomplete proteins with adverse effects on the cell – Stalled ribosomes out of action – Unable to participate in further protein synthesis 18-46 Non-Stop mRNAs • Prokaryotes deal with non-stop mRNAs by tmRNAmediated ribosome rescue – Alanyl-tmRNA resembles alanyl-tRNA – Binds to vacant A site of a ribosome stalled on a non-stop mRNA – Donates its alanine to the stalled polypeptide • Ribosome shifts to translating an ORF on the tmRNA (transfer-messenger RNA) – Adds another 9 amino acids to the polypeptide before terminating – Extra amino acids target the polypeptide for destruction – Nuclease destroys non-stop mRNA 18-47 Non-Stop mRNAs • Prokaryotes deal with non-stop mRNAs by tmRNA-mediated ribosome rescue – tmRNA are about 300 nt long – 5’- and 3’-ends come together to form a tRNA-like domain (TLD) resembling a tRNA 18-48 Eukaryotic Aberrant Termination • Eukaryotes do not have tmRNA • Eukaryotic ribosomes stalled at the end of the poly(A) tail contain 0 – 3 nt of poly(A) tail – This stalled ribosome state is recognized by carboxylterminal domain of a protein called Ski7p – Ski7p also associates tightly with cytoplasmic exosome, cousin of nuclear exosome – Non-stop mRNA recruit Ski7p-exosome complex to the vacant A site – Ski complex is recruited to the A site • Exosome, positioned just at the end of non-stop mRNA, degrades that RNA • Aberrant polypeptide is presumably destroyed 18-49 Exosome-Mediated Degradation • This stalled ribosome state is recognized by carboxylterminal domain of a protein called Ski7p • Ski7p also associates tightly with cytoplasmic exosome, cousin of nuclear exosome • Non-stop mRNA recruit Ski7p-exosome complex to the vacant A site • Ski complex is recruited to the A site 18-50 Premature Termination • Eukaryotes deal with premature termination codons by 2 mechanisms: – NMD (nonsense-mediated mRNA decay) • Mammalian cells use a downstream destabilizing element • Yeast cells appear to recognize a premature stop codon – NAS (nonsense-associated altered splicing) • Senses a stop codon in the middle of a reading frame • Changes the splicing pattern so premature stop codon is spliced out of mature mRNA – Both mechanisms require Upf1 18-51 Mammalian NMD NMD in mammalian cells involves a downstream destabilizing element • Upf1 • Upf2 – Bind to mRNA at exon-exon junction that measures distance to a stop codon – Codon far enough upstream • Looks like a stop codon • Activates downstream destabilizing element to degrade mRNA 18-52 Yeast NMD • Yeast cells appear to recognize a premature stop codon by the absence of a normal 3’-UTR or poly (A) nearby • Ribosome stopping at premature stop codon moves to an upstream AUG • This may mark the mRNA for destruction 18-53 NAS and NMD Models 18-54 Use of Stop Codons to Insert Unusual Amino Acids Unusual amino acids are incorporated into growing polypeptides in response to termination codons – Selenocysteine uses a special tRNA • Anticodon for UGA codon • Charged with serine then converted to selenocysteine • Selenocysteyl-tRNA escorted to ribosome by special EF-Tu – Pyrrolysine uses a special tRNA synthetase that joins preformed pyrrolysine with a special tRNA having an anticodon recognizing UAG 18-55 18.5 Posttranslation • Translation events do not end with termination – Proteins must fold properly – Ribosomes need to be released from mRNA and engage in further translation rounds • Folding is actually a cotranslational event occurring as nascent polypeptide is being made 18-56 Folding Nascent Proteins • Most newly-made polypeptides do not fold properly alone – Polypeptides require folding help from molecular chaperones – E. coli cells use a trigger factor • Associates with the large ribosomal subunit • Catches the nascent polypeptide emerging from ribosomal exit tunnel in a hydrophobic basket to protect from water – Archaea and eukaryotes lack trigger factor, use freestanding chaperones 18-57 Release of Ribosomes from mRNA • Ribosomes do not release from mRNA spontaneously after termination • Help is required from ribosome recycling factor (RRF) and EF-G – RRF resembles a tRNA • Binds to ribosome A site • Uses a position not normally taken by a tRNA – Collaborates with EF-G in releasing either 50S ribosome subunit or whole ribosome 18-58