Transcript Inquiry into Life Twelfth Edition

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 aminocarboxyl
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