Initiation of Translation

1
Initiation of Translation
1. Some basics about translation
2. The process of initiation

• Prokaryotes
• Eukaryotes

3. Control of initiation

• Prokaryotes
• Eukaryotes

2
Small RNAs Regulate Translation in Prokaryotes
More than 40 sRNAs identified in E. coli
Only a handful have been characterized, but
these bind by base-pairing to mRNA targets and
regulate translation
DsrA RNA forms base pairs with rpoS mRNA, which
exposes Shine-Dalgarno sequence
rpoS encodes a stationary phase sigma factor
Some other sRNAs are known to repress
translation intead of activating
Fig. 17.35
3
Riboswitches A Common Prokaryotic Mechanism of
Translation Regulation
Discovered by Breaker and colleagues (2002)
Shown is thiamine pyrophosphate (TPP)
riboswitch. Binding of TPP represses translation
of mRNAs that encode enzymes involved in TPP
synthesis
Biochemical studies performed to show clearly
that TPP binds to RNA element, inducing
conformational change that blocks Shine-Dalgarno
sequence
Riboswitches are ubiquitous in prokaryotes.
In addition to substances like vitamins, ligands
are - amino acids - nucleobases (guanine
and adenine) - sugars - metal ions
Fig. 17.36
4
Initiation of Translation
1. Some basics about translation
2. The process of initiation

• Prokaryotes
• Eukaryotes

3. Control of initiation

• Prokaryotes
• Eukaryotes

5
Phosphorylation of Initiation Factor eIF2?
Used extensively in reticulocytes, which
basically make only hemoglobin
If cell is starved for iron, heme-controlled
repressor phosphorylates one of the subunits of
eIF2, known as eIF2?
Phosphorylated eIF2 binds eIF2B tightly, which
sequesters eIF2B such that it is unable to
promote GDP/GTP exchange
This leads to global repression of translation
Fig. 17.37
6
Regulation by an RNA Element
Munro, Klausner, and colleagues demonstrated
repression of translation by RNA sequence (1987)
Repressor protein binds IRE element in 5 UTR
until removed by Fe2
When Fe2 level is high, increased translation
of protein ferritin allows for increased iron
storage.
Assayed using chloramphenicol acetyltransferase
(CAT) reporter gene and monitoring acetylation of
chloramphenicol using TLC system
Lacked IRE
Fig. 17.45
7
More Recent Structural Work Has Further
Elucidated Mechanism
NMR showed structural changes in element of RNA
in 5 UTR upon binding of repressor IRP1 (also
shown to be aconitase).
Stem structure is only 20 nt from 5 end,
suggesting that repression is mediated by
preventing the 40S subunit from binding.
Repressor protein, aconitase, also binds Fe-S
cluster. Binding of this cluster leads to a
conformational change which prevents binding to
IRE
Leipuviene and Theil, Cell. Mol. Life Sci. (2007)
64, 2945-2955
8
miRNA A Major Pathway For Translational
Regulation in Eukaryotes
We will hear much more about micro RNAs
(miRNAs) in two weeks
Fig. 16.45
9
Key Points
1. Prokaryotic and eukaryotic translation
initiation have some common features. A charged
initiator tRNA forms a complex with the small
subunit that recognizes a start codon, followed
by joining of the large subunit, GTP hydrolysis,
and ultimately initiation.
2. Eukaryotic initiation has unique features
most is cap-dependent and occurs by scanning, the
mRNA circularizes, and there are more factors.
Some mRNAs (especially viral) use IRES elements
3. Prokaryotes and eukaryotes have varied
mechanisms for control of translation. These make
extensive use of RNA-RNA interactions and RNA
structures.
10
The Process of Translation Elongation and
Termination
1. Determining the genetic code
2. Translation elongation
3. Translation termination
4. Rescuing translation when things dont work
11
Frameshift Mutations and Evidence for a Triplet
Code
Fig. 18.3
12
Proof of a Triplet Code
Khorana and colleagues were able to synthesize
RNAs with repeating sequences
a repeating dinucleotide produced in
translation a repeating di-peptide a repeating
trinucleotide produced a mixture of three
homopeptides a repeating tetranucleotide
producted a tetrapeptide
Fig. 18.4
13
A tRNA Binding Assay to Help Break the Code
Different aminoacylated tRNAs bound to
ribosomes in the presence of different
tri-nucleotides (Khorana, 1968)
Fig. 18.6
Khorana shared Nobel Prize in Medicine (1968)
for work on translation and the genetic code
Fig. 18.5
14
Base-pairing Between Codon and Anti-codon
Fewer than 60 tRNAs needed because of wobble
pairing
There are also tRNAs that have different
anticodon loops but are charged with the same
amino acid
Fig. 18.7
Fig. 18.8
15
The Process of Translation Elongation and
Termination
1. Determining the genetic code
2. Translation elongation

• binding of aminoacylated tRNA to the A site
• formation of new peptide bond
• translocation of the peptidyl-tRNA to the P site

3. Translation termination
4. Rescuing translation when things dont work
16
Overview of Elongation
Fig. 18.10
17
1st step Initiation
T. Terry, U. Conn
18
2nd step Elongation
T. Terry, U. Conn
19
Last step Termination
T. Terry, U. Conn
20
Puromycin An Antibiotic and Valuable
Experimental Tool
Fig. 18.11
21
Aminoacyl-tRNA Binding to the A Site
Fig. 18.14
22
Formation of the Ternary Complex of GTP,
Aminoacyl-tRNA, and EF-Tu
Joanne Ravel (1968)
 Sephadex G100 gel filtration
 Both GTP and Phe-tRNAPhe were present in larger
complex
 EF-Tu had not yet been fractionated it was
just called EF-T, a mixture of EF-Tu and EF-Ts
Fig. 18.17
23
EF-Ts Is Only Necessary For Ternary Complex
Formation When Starting From EF-Tu-GDP
Binding monitored by nitrocellulose filter
binding of radioactive aminoacyl-tRNA
Fig. 18.18
24
How Does a Guanine Nucleotide Exchange Factor
Work?
Fig. 18.26
25
Aminoacyl-tRNA Binding to the A Site
Fig. 18.14
26
One Last Chance To Reject the Amino Acid Before
It Gets Incorporated
Irreversible GTP hydrolysis step allows for an
additional proofreading step
Fig. 18.20
27
Conformational Changes of the Decoding Center
Fig. 19.9
Fig. 19.8
28
Conformational Changes of the Decoding Center
A1492 and A1493 form A-minor interactions with
the anticodon-mRNA base pairs
First discovered because antibiotic paromomycin
induces flipping out of these bases even in the
absence of mRNA or tRNA
Effect of paromomycin is to decrease
specificity, apparently by decreasing the
energetic penalty for incorrect bases and
therefore decreasing the ability to proofread
Fig. 19.13
29
Conformational Changes of the Decoding Center
A-minor interactions between A1493 and
codon-anticodon
Contacts are formed between A1493 and ribose of
each nucleotide in the codon-anticodon base pair
(type I A-minor interaction)
Fig. 19.12
30
Puromycin Reaction To Assay Peptide Bond Formation
Fig. 18.21
31
Peptidyl-transferase Activity of Nearly-fully
Deproteinized Ribosomes
Noller and colleagues (1992)
Significant activity detected for ribosomes or
50S subunits even with more than 90 of protein
removed
Strongly suggested that RNA is catalytic
component of ribosome
Fig. 18.23
32
The Chemistry of Peptidyl Transfer
No specifically bound Mg2 ions observed in
reaction center
No individual nucleotide substitutions within
23S rRNA give large effects on chemical step
Deletion of 2-OH of P site tRNA effectively
abolishes catalysis
Schmeing et al, Mol Cell (2005), 437-448
33
Next step Translocation
Translocation assayed by release of deacylated
tRNA or by puromycin assay (release of labeled
Phe)
Initially concluded that GTP binding, but not
hydrolysis, was necessary for translocation.
Therefore, translocation was thought to come
before GTP hydrolysis.
Fig. 18.24
34
Kinetics of Translocation
Wintermeyer and colleagues, 1997
Signal for translocation from a fluorescent
peptidyl-tRNA in the A site
GTP hydrolysis monitored separately with
radiolabeled GTP
GTP is hydrolyzed faster than translocation
occurs, so hydrolysis precedes translocation
But translocation can still occur, albeit more
slowly, without GTP hydrolysis
Translocation almost certainly involves complex
movements of the ribosomal subunits that remain
poorly understood
Fig. 18.25
35
The Process of Translation Elongation and
Termination
1. Determining the genetic code
2. Translation elongation

• binding of aminoacylated tRNA to the A site
• formation of new peptide bond
• translocation of the peptidyl-tRNA to the P site

3. Translation termination
4. Rescuing translation when things dont work
36
Early Experiments of Nonsense Mutations (Amber
here) and Suppressor Strains
Fig. 18.28
37
Revertants Were Used To Deduce the Identity of
the Amber Codon
Revertants gave incorporation of six different
amino acids
It was easier to sequence polypeptides than DNA!
Fig. 18.29
38
Mechanism of Suppression
Mutation in tRNA allows recognition of stop
codon and incorporation of amino acid
More than one tRNA be mutated to become a
suppressor
Fig. 18.30
Fig. 18.31
39
Assay For a Release Factor
Ribosome stalled with labeled peptide
Different ribosomal fractions then added to
find one that would allow release of labeled
peptide
Fig. 18.32
40
A Simpler Assay For Release Factors
fMet-tRNAfMet loaded in P site bound to AUG
Tri-nucleotide corresponding to stop codon added
Addition of ribosome fractions containing
appropriate release factor would allow release of
labeled fMet into solution
41
Structure of Release Factor Resembles tRNA
42
The Process of Translation Elongation and
Termination
1. Determining the genetic code
2. Translation elongation

• binding of aminoacylated tRNA to the A site
• formation of new peptide bond
• translocation of the peptidyl-tRNA to the P site

3. Translation termination
4. Rescuing translation when things dont work
43
Bacterial tmRNA An Elegant Solution to Problems
In Translation
Some aberrant RNAs lack termination codons,
causing ribosomes to read to the ene and then
stall
tmRNA provides three services to the cell
1. It allows recycling of stalled ribosomes 2.
It targets the aberrant RNA for degradation 3.
It targets the aberrant peptide for degradation
44
Mechanism of tmRNA-mediated Release of Non-stop
mRNA
45
Key Points
1. All organisms use essentially the same genetic
code three nucleotides comprise a codon, which
specifies an amino acid or a termination.
2. Translation elongation consists of cycles of
1) multi-step binding of aminoacyl tRNA in a
ternary complex with EF-Tu and GTP 2) peptidyl
transfer, catalyzed by the 50S subunit and 3)
translocation, which is greatly facilitated by
EF-G and GTP.
3. Termination is mediated by the appropriate
stop codons and release factors, which recognize
them. In the absence of termination on aberrant
RNAs, bacterial cells have evolved the tmRNA to
rescue stalled ribosomes and target the RNA and
protein for degradation.
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