Transcript The Mechanism of Translation II

The Mechanism of Translation II:
Elongation and Termination
Chapter 18
Direction of Polypeptide Synthesis and
mRNA Translation
• Messenger RNAs are read in the 5’3’
direction
• Proteins are made in the aminocarboxyl
direction - means that the amino terminal
amino acid is added first
The Genetic Code
• The term genetic code refers to the set of 3base code words (codons) in mRNA that
represent the 20 amino acids in proteins
• 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 codons
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
Coding Properties of Synthetic
mRNAs
The Genetic Code
• There are 64 codons
– 3 are stop signals
– Remainder code for
amino acids
– The genetic code is
highly degenerate
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 nonWatson-Crick base pair with the anticodon
– Wobble allows same aminoacyl-tRNA to pair with
more than one codon
Compare standard Watson-Crick base pairing with
wobble base pairs
• Wobble pairs are:
– G-U
– I-A
Deviations from “Universal”
Genetic Code
The (Almost) universal code
• Genetic code is NOT strictly universal
• Transitions and Transversions
• 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
Elongation mechanism in E.coli
Elongation takes place in three steps:
1. EF-Tu with GTP binds
aminoacyl-tRNA to the
ribosomal A site (empty –
based on second codon).
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
Elongation mechanism in E.coli Translocation
• EF-G with GTP translocates
the growing peptidyl-tRNA
with its mRNA codon to the
P site
- The deacylated tRNA in P
site leaves ribosome via E
site.
- The dipeptidyl-tRNA in A
site along with its
corresponding codon moves
into P site
- Steps keep on repeating
Protein Factors and Peptide Bond
Formation
• One factor is T- transfer
– It transfers aminoacyl-tRNAs to the ribosome
– has 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
Elongation Step 2
• One 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
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
Elongation Step 3
• When peptidyl transferase has worked:
– Ribosome has peptidyl-tRNA in the A site
– Deacylated tRNA in the P site
• Translocation - moves mRNA and peptidyltRNA 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
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
Protein-Synthesizing Machinery
• Two steps achieve accuracy:
– Gets rid of ternary complexes bearing wrong aminoacyltRNA 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 codon-anticodon
base pairing to ensure dissociation occurs more
rapidly than either GTP hydrolysis or peptide bond
formation
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
Three-Nucleotide Movement
Each translocation event
moves the mRNA on
codon length, or 3 nt
through the ribosome
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
Termination
• Elongation cycle repeats over and over
– Adds amino acids one at a time
– Grows the polypeptide product
• Finally ribosome encounters a stop codon
UAG, UAA, UGA
– Stop codon signals time for last step
– Translation last step is termination
Termination Mutations
• 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
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
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
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
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 tRNAlike domain (TLD)
resembling a tRNA
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
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-Mediated Degradation
• Exosome, positioned just at the end of non-stop mRNA,
degrades that RNA
• Aberrant polypeptide is presumably destroyed
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
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
Release of Ribosomes from mRNA
• 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
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