Molecular BiologyFifth Edition

Chapter 18The Mechanism of

Translation II: Elongation and

Termination

Lecture PowerPoint to accompany

Robert F. Weaver

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

18-2

Elongation and Termination• Elongation is very similar in bacteria and

eukaryotes• Consider the following fundamental

questions:• In what direction is a polypeptide

synthesized?• In what direction does the ribosome read

the RNA?• What is the nature of the genetic code that

dictates which amino acids will be incorporated in response to the mRNA?

18-3

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

18-4

Strategy to determine the direction of Translation

18-5

18.2 The Genetic Code

• The term genetic code refers to the set of 3-base code words or 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

18-6

Nonoverlapping Codons

• Codons are nonoverlapping in the message or mRNA

• 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 codons

18-7

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

18-8

Frameshift MutationsFrameshift mutations • Translation starts AUGCAGCCAACG

• Insert an extra base 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 leftCode 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 AUGZCAGZCCAZACGZ

• Insert an extra base AUXGZCAGZCCAZACGZ

– First codon wrong, all others separated by Z, translated normally

18-9

Frameshift Mutation Sequences

18-10

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

18-11

Coding Properties of Synthetic mRNAs

18-12

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

18-13

The Genetic Code

18-14

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

18-15

Superwobble Hypothesis

• According to the wobble hypothesis, a cell should be able to get by with only 31 tRNAs to read all 64 codons

• Human and plant mitochondria contain less than 31 tRNAs

• The superwobble hypothesis holds that a single tRNA with a U in its wobble position can, in certain circumstances, recognize codons ending in ay of the 4 bases

• Tested by Ralph Block and colleagues in tobacco plastids

18-16

Wobble Base Pairs

• Compare standard Watson-Crick base pairing with wobble base pairs

• Wobble pairs are:– G-U– I-A

18-17

Wobble Position

18-18

The (Almost) Universal Code

• Genetic code is NOT strictly universal• Certain eukaryotic nuclei and mitochondria along

with at least one bacterium have altered code– 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

18-19

Deviations from “Universal” Genetic Code

18-20

18.3 The Elongation Cycle

Elongation takes place in a three step cycle:1. EF-Tu with GTP binds aminoacyl-tRNA to

the ribosomal A site2. Peptidyl transferase forms a peptide bond

between peptide in P site and newly arrived aminoacyl-tRNA in the A siteLengthens 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

18-21

Elongation in Translation

18-22

A Three-Site Model of the Ribosome

• The existence of the A and P sites was originally based on experiments with the antibtiotic puromycin – Resembles an aminoacyl-tRNA– Can bind to the A site– Couple with the peptide in the P site– Release it as peptidyl puromycin

18-23

A Three-Site Model of the Ribosome

• 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

• Terminology:• E site - Exit• P site - Peptidyl• A site - Aminoacyl

18-24

Puromycin Structure and Activity

18-25

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

18-26

Elongation Step 1Binding 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

18-27

Aminoacyl-tRNA binding to ribosome A Site

18-28

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

18-29

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

aminoacyl-tRNA in the proofreading step before wrong amino acid is incorporated into growing protein chain

• Steps rely on weakness of incorrect codon-anticodon base pairing to ensure dissociation occurs more rapidly than either GTP hydrolysis or peptide bond formation

18-30

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

18-31

Elongation Step 2

• Once the initiation factors and EF-Tu have done their jobs, the ribosome has fMet-tRNA 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

18-32

Assay for Peptidyl Transferase

18-33

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

18-34

Elongation Step 3

• Once peptidyl transferase has done its job:– 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

18-35

Translocation - Movement of Nucleotides

Each translocation event moves the mRNA on codon length, or 3 nt through the ribosome

18-36

Role of GTP and EF-G

• GTP and EF-G are necessary for translocation although translocation activity appears to be inherent in the ribosome and can be expressed without EF-G and GTP in vitro

• GTP hydrolysis precedes translocation and significantly accelerates it

• New round of elongation occurs if:– EF-G is released from the ribosome, which

depends on GTP hydrolysis

18-37

G Proteins 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

18-38

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

18-39

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

18-40

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

18-41

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

18-42

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

18-43

Amber Mutation Effects in a Fused Gene

18-44

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

18-45

Termination Mutations

18-46

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

18-47

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

18-48

Release Factor Assay

18-49

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

18-50

Non-Stop mRNAs

• Prokaryotes deal with non-stop mRNAs by tmRNA-mediated 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

18-51

Structure of tmRNAs

• 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

18-52

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 carboxyl-

terminal 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

18-53

Exosome-Mediated Degradation

• This stalled ribosome state is recognized by carboxyl-terminal 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

18-54

Premature Termination

• Eukaryotes deal with premature termination codons by 2 mechanisms:– NMD (nonsense-mediated mRNA decay)

• Mammalian cells rely on the ribosome to measure the distance between the stop codon and the EJC - if it is too long the mRNA is destroyed

• 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

18-55

NAS and NMD Models

18-56

No-go Decay (NGD)

• Another kind of mRNA decay which begins with an endonucleolytic cleavage near the stalled ribosome

• It provides another potential means of post-transcriptional control by selective degradation of mRNAs

18-57

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

18-58

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

18-59

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

18-60

Release of Ribosomes from mRNA

• Ribosomes do not release from mRNA spontaneously after termination

• Eukaryotic ribosomes are released by eIF3, aided by eIF1, eIFA and eIF3j

• Prokaryotic ribosomes require help 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 the whole ribosome