Lecture Power Point to accompany Molecular Biology Fourth

Lecture Power. Point to accompany Molecular Biology Fourth Edition Robert F. Weaver Chapter 18 The Mechanism of Translation II: Elongation and Termination Copyright © The Mc. Graw-Hill Companies, Inc. Permission required for reproduction or display.

18. 1 Direction of Polypeptide Synthesis and m. RNA 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 amino carboxyl direction • This means that the amino terminal amino acid is added first 2

Strategy to Determine Direction of Translation 3

18. 2 The Genetic Code • The term genetic code refers to the set of 3 -base code words (codons) in m. RNA that represent the 20 amino acids in proteins • Basic questions were answered about translation in the process of “breaking” the genetic code 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 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 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 normally 7

Frameshift Mutation Sequences 8

The Triplet Code • The genetic code is a set of three-base code words, or codons – In m. RNA, 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 m. RNA is part of a codon 9

Coding Properties of Synthetic m. RNAs 10

Breaking the Code • The genetic code was broken – Using: • Synthetic messengers • Synthetic trinucleotides – Then observing: • Polypeptides synthesized • Aminoacyl-t. RNAs bound to ribosomes • There are 64 codons – 3 are stop signals – Remainder code for amino acids – The genetic code is highly degenerate 11

The Genetic Code 12

Unusual Base Pairs Between Codon and Anticodon Degeneracy of genetic code is accommodated by: – Isoaccepting species of t. RNA: bind same amino acid, but recognize different codons – Wobble, the 3 rd 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-t. RNA to pair with more than one codon 13

Wobble Base Pairs • Compare standard Watson-Crick base pairing with wobble base pairs • Wobble pairs are: – G-U – I-A 14

Wobble Position 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 16

Deviations from “Universal” Genetic Code 17

18. 3 The Elongation Mechanism Elongation takes place in three steps: 1. EF-Tu with GTP binds aminoacyl-t. RNA to the ribosomal A site 2. Peptidyl transferase forms a peptide bond between peptide in P site and newly arrived aminoacyl-t. RNA 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-t. RNA with its m. RNA codon to the P site 18

Elongation in Translation 19

A Three-Site Model of the Ribosome • Puromycin – – Resembles an aminoacyl-t. RNA Can bind to the A site Couple with the peptide in the P site Release it as peptidyl puromycin • If peptidyl-t. RNA 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) • 3 rd site (E) for deacylated t. RNA bind to E site as exits ribosome 20

Puromycin Structure and Activity 21

Protein Factors and Peptide Bond Formation • One factor is T, transfer – It transfers aminoacyl-t. RNAs 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 22

Elongation Step 1 Binding aminoacyl-t. RNA to A site of ribosome • Ternary complex formed from: – – – EF-Tu Aminoacyl-t. RNA GTP • Delivers aminoacyl-t. RNA 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-t. RNA reconstitutes ternary complex for another round of translation elongation 23

Aminoacyl-t. RNA Binding to Ribosome A Site 24

Proofreading • Protein synthesis accuracy comes from charging t. RNAs with correct amino acids • Proofreading is correcting translation by rejecting an incorrect aminoacyl-t. RNA before it can donate its amino acid • Protein-synthesizing machinery achieves accuracy during elongation in two steps 25

Protein-Synthesizing Machinery • Two steps achieve accuracy: – Gets rid of ternary complexes bearing wrong aminoacyl-t. RNA before GTP hydrolysis – If this screen fails, still eliminate incorrect aminoacylt. RNA 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 26

Proofreading Balance • Balance between speed and accuracy of translation is delicate – If peptide bond formation goes too fast • Incorrect aminoacyl-t. RNAs 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 27

Elongation Step 2 • One the initiation factors and EF-Tu have done their jobs, the ribosome has f. Mett. RNA in the P site and aminoacyl-t. RNA 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 28

Assay for Peptidyl Transferase 29

Peptide Bond Formation • The peptidyl transferase resides on the 50 S ribosomal particle • Minimum components necessary for activity are 23 S r. RNA and proteins L 2 and L 3 • 23 S r. RNA is at the catalytic center of peptidyl transferase 30

Elongation Step 3 • When peptidyl transferase has worked: – Ribosome has peptidyl-t. RNA in the A site – Deacylated t. RNA in the P site • Translocation, next step, moves m. RNA and peptidyl-t. RNA one codon’s length through the ribosome – Places peptidyl-t. RNA in the P site – Ejects the deacylated t. RNA – Process requires elongation factor EF-G which hydrolyzes GTP after translocation is complete 31

Three-Nucleotide Movement Each translocation event moves the m. RNA on codon length, or 3 nt through the ribosome 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 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 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 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 36

Structures of EF-Tu and EF-G • Three-dimensional shapes determined by x-ray crystallography: – EF-Tu-t. RNA-GDPNP ternary complex – EF-G-GDP binary complex • As predicted, the shapes are very similar 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 38

Termination Codons • Three codons are the natural stop signals at the ends of coding regions in m. RNA – UAG – UAA – UGA • Mutations can create termination codons within an m. RNA causing premature termination of translation – Amber mutation creates UAG – Ochre mutation creates UAA – Opal mutation creates UGA 39

Amber Mutation Effects in a Fused Gene 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 41

Termination Mutations 42

Stop Codon Suppression • Most suppressor t. RNAs have altered anticodons: – Recognize stop codons – Prevent termination by inserting an amino acid – Allow ribosome to move on to the next codon 43

Release Factors • Prokaryotic translation termination is mediated by 3 factors: – RF 1 recognizes UAA and UAG – RF 2 recognizes UAA and UGA – RF 3 is a GTP-binding protein facilitating binding of RF 1 and RF 2 to the ribosome • Eukaryotes has 2 release factors: – e. RF 1 recognizes all 3 termination codons – e. RF 3 is a ribosome-dependent GTPase helping e. RF 1 release the finished polypeptide 44

Release Factor Assays 45

Dealing with Aberrant Termination • Two kinds of aberrant m. RNAs can lead to aberrant termination – Nonsense mutations can occur that cause premature termination – Some m. RNAs (non-stop m. RNAs) lack termination codons • Synthesis of m. RNA was aborted upstream of termination codon • Ribosomes translate through non-stop m. RNAs 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 46

Non-Stop m. RNAs • Prokaryotes deal with non-stop m. RNAs by tm. RNAmediated ribosome rescue – Alanyl-tm. RNA resembles alanyl-t. RNA – Binds to vacant A site of a ribosome stalled on a non-stop m. RNA – Donates its alanine to the stalled polypeptide • Ribosome shifts to translating an ORF on the tm. RNA (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 m. RNA 47

Non-Stop m. RNAs • Prokaryotes deal with non-stop m. RNAs by tm. RNA-mediated ribosome rescue – tm. RNA are about 300 nt long – 5’- and 3’-ends come together to form a t. RNA-like domain (TLD) resembling a t. RNA 48

Eukaryotic Aberrant Termination • Eukaryotes do not have tm. RNA • 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 Ski 7 p – Ski 7 p also associates tightly with cytoplasmic exosome, cousin of nuclear exosome – Non-stop m. RNA recruit Ski 7 p-exosome complex to the vacant A site – Ski complex is recruited to the A site • Exosome, positioned just at the end of non-stop m. RNA, degrades that RNA • Aberrant polypeptide is presumably destroyed 49

Exosome-Mediated Degradation • This stalled ribosome state is recognized by carboxylterminal domain of a protein called Ski 7 p • Ski 7 p also associates tightly with cytoplasmic exosome, cousin of nuclear exosome • Non-stop m. RNA recruit Ski 7 p-exosome complex to the vacant A site • Ski complex is recruited to the A site 50

Premature Termination • Eukaryotes deal with premature termination codons by 2 mechanisms: – NMD (nonsense-mediated m. RNA 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 m. RNA – Both mechanisms require Upf 1 51

Mammalian NMD in mammalian cells involves a downstream destabilizing element • Upf 1 • Upf 2 – Bind to m. RNA 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 m. RNA 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 m. RNA for destruction 53

NAS and NMD Models 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 t. RNA • Anticodon for UGA codon • Charged with serine then converted to selenocysteine • Selenocysteyl-t. RNA escorted to ribosome by special EF-Tu – Pyrrolysine uses a special t. RNA synthetase that joins preformed pyrrolysine with a special t. RNA having an anticodon recognizing UAG 55

18. 5 Posttranslation • Translation events do not end with termination – Proteins must fold properly – Ribosomes need to be released from m. RNA and engage in further translation rounds • Folding is actually a cotranslational event occurring as nascent polypeptide is being made 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 57

Release of Ribosomes from m. RNA • Ribosomes do not release from m. RNA spontaneously after termination • Help is required from ribosome recycling factor (RRF) and EF-G – RRF resembles a t. RNA • Binds to ribosome A site • Uses a position not normally taken by a t. RNA – Collaborates with EF-G in releasing either 50 S ribosome subunit or whole ribosome 58