Transcript Translational Initiation in Eukaryotes

(Lost in) Translation
1. Briefly review prokaryotic machinery
2. Initiation in Eukaryotes
3. Where in the world is Peptidyl
Transferase?
4. tRNA charging: The second code
1968 Nobel Prize in Physiology & Medicine
(for deciphering the genetic code)
“Triumph of the Chemists”
H.G. Khorana
R. Holley
M. Nirenberg
Used a cell-free protein synthesis system from E. coli, programmed it with
natural and synthetic RNAs of defined sequence, and determined the
sequence of the peptides produced.
The cell-free system
1. S-30 fraction
– ribosomes, tRNAs, tRNA synthetases, other
soluble protein factors
2. 20 amino acids
3. GTP & ATP
4. Energy generating system
– to keep producing ATP and limit [ADP]
– PEP + pyruvate kinase
PEP + ADP + Pi  pyruvate + ATP
5. Mg2+ and K+ (NH4+)
Translation Machinery in Prokaryotes
(for comparing with Eukaryotes)
• Ribosomes:
-70S (composed of L (50S) and S (30S)
subunits)
-contain 23S (L), 16S (S), and 5S (L) rRNAs
-each subunit (L and S) contains ~30 proteins
• Initiation factors: if1, if2, if3
• Elongation factors: ef-Tu, ef-Ts, and G
• Termination (release) factor(s): Rf1 and Rf2
• Translation is initiated with fmet (N-formylated
methionine).
How is right AUG selected for
translation in Prokaryotes?
1.
Many mRNAs contain a sequence preceding the
start codon that base-pairs with the 3'-end of 16S
rRNA (Shine-Dalgarno sequence)
S-D
start
5'----GGAGG-------AUG-----3’ mRNA
3'----CCUCC--------5'
16S rRNA
2.
3.
4.
Function: helps position mRNA in ribosome.
The AUG itself is also very important
There is a S-D independent mode of translation
initiation in E. coli
Translate internal ORFs of polycistronic mRNAs
Translational Initiation in
Eukaryotes
• Begins with methionine that is not formylated
• tRNA (tRNAiMet) different from the one that is
used for internal methionine codons
• Translation start determined by the AUG and
surrounding sequence
• Translation start site also affected by RNA
structure at the 5’ end of the mRNA
Scanning Model of Initiation
• Proposed by M. Kozak
• Small subunit of ribosome (+ initiation factors,
GTP and tRNAiMet) binds to the 5’ Cap, and
scans along the mRNA until the first AUG
• Translation starts at the first AUG
• Model seems to work for most mRNAs
Scanning (or Kozak) Model for
Translation Initiation in Eukaryotes
ATP
Fig. 17.16
Apparent Exceptions to the Scanning
Model?
• Translation of some mRNAs (5-10%) doesn’t
start at first AUG (ribosome skips one or
more AUGs)
• Comparative sequence analysis of these
mRNAs revealed the following consensus
sequence at the AUG that is used:
-5 -4 -3 -2 -1 +1 +2 +3 +4
CCRCCAUGG
R=purine
• Positions -3 and +4 are particularly important,
based on mutagenesis studies
Effect of the context of an upstream “barrier” ATG on
initiation of preproinsulin mRNA.
Fig. 17.18
proinsulin
Conclusion: When the upstream AUG was in a weak context (like
F9), then the downstream one is used. Or, put another way,
the first AUG in the right context is used.
Upstream ATG is an ineffective barrier if followed by a
Stop codon.
Stop codon
In some mRNAs, the first
ATG is in a favorable
context, but is still not
used. Kozak noted that
there was usually a Stop
codon in between the start
codons in these mRNAs. So
she engineered such a
situation in the preproinsulin
mRNA and tested its affect
on translation.
Result: Translation was good at the
downstream ATG as long as it was in a good
context.
Fig. 17.23
2nd ed.
Conclusions
• An upstream AUG does not interfere if it’s
context (-3,+4) is poor, or if it is followed
quickly by an in-frame Stop codon.
- In the latter case, it may be that the
ribosomes don’t fall off the mRNA after
translating such a short ORF.
- In natural mRNAs, upstream ORFs are
very short, unless they have a regulatory
role.
Is the first “good” AUG really
favored? Effect of Repeated
Initiation Sequences (replicas)
AUG
AUG
AUG
Translation started mainly at the first AUG.
Fig. 17.19
Effect of RNA Secondary Structure in the
5’ UTR (Leader)
Poorly translated
Translated well
Not translated
Trans. well
Adapted from Fig. 17.20
Conclusions
• Secondary structure (hairpin) at very 5’ end of
RNA can prevent 40S subunit from binding
• Scanning ribosomes can melt out some
hairpins ( ΔG= -30 kcal/mole), but not highly
stable ones ( ΔG= -62 kcal/mole)
• Initiator tRNA (tRNAiMet) also important in
recognizing AUG
– (yeast) Anticodon of tRNAiMet changed to UCC,
translation started at first “good” AGG in his4
mRNA (Fig. 17.21).
Summary of translation initiation in
Eukaryotes.
Resists binding to 60S subunit
Fig. 17.22
Initiation Factors (except eIF-4)
eIF-1(and 1A): promotes scanning
*eIF-2: binds tRNAiMet to 40S subunit, requires
GTP (which gets hydrolyzed to GDP)
eIF-2B: catalyzes exchange of GTP for GDP on
eIF-2
*eIF-3: binds to 40S subunit, prevents 60S subunit
from binding to it
eIF-5: stimulates 60S subunit binding to the 48S
pre-initiation complex
*eIF-6: binds to 60S subunit, helps prevent 40S
subunit from binding to it
* prokaryotic counterpart
eIF4 (eIF4F)
eIF4F
Originally isolated based on its ability to
bind the Cap-nucleotide 7MeGTP.
Composed of 3 subunits, a 24-kDa
protein that binds the Cap, and 2
others that stabilize the complex and
have other roles:
1. eIF4G - versatile adaptor
2. eIF4A - RNA helicase
3. eIF4E - binds the Cap
Fig. 17.25
eIF4A and eIF4B
eIF4A
• also exists outside of the
eIF4F complex
• contains a DEAD motif
(aspartate-glutamate-alanineaspartate) characteristic of
RNA helicases
• RNA helicase activity was
demonstrated (right panel)
and found to require ATP and
to be stimulated by another
protein, eIF4B
eIF4B
• binds RNA, stimulates eIF-4A
Role in translation: Unwind hairpins in the 5’ UTRs
17.26
eIF4G – helps recruit 40S subunit to mRNA; can
interact with eIF4E, eIF4A, eIF3, and poly-A binding
protein (Pab1); may be responsible for the
synergistic effect of Cap and polyA-tail on
translation.
Similar to 17.27c
Why interact with both Cap and polyA-tail?
Observation: Some viral mRNAs (such as Polio virus)
are not capped, yet are preferentially translated. Some
are also translated via internal ribosome entry sites
(IRES) (apparently without scanning to them).
Mechanism: Viral protease clips off N-terminus of
eIF4G, so it can’t bind eIF4E. eIF4G binds a viral
protein (X), that binds to the IRES, promoting translation
of the uncapped viral mRNAs.
17.27
eIF1 & eIF1A
• Genes essential in yeast
• Needed for the 40S subunit-particle to
scan more than a few nucleotides
from the Cap and form the 48S
complex
• Also dissociate improperly formed
complexes between the 40S subunit
and mRNA
Toe-printing assay for determining where the leading
edge of a ribosome (or ribosomal subunit) is on a
mRNA
Fig. 17.31
RESULTS:
formation of
Complex II, which
is the toe print of
the 40S subunit
that has scanned
to the AUG, is
obtained only
when eIF1 +
eIF1A, or a
fraction containing
them (50-70%
A.S.), was added.
eIF1+eIF1A also convert Complex I into Complex II when
added after Complex I has formed (lane 8).
Fig. 17.32