(Lost in) Translation

1
(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

2
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.
3
The cell-free system

• S-30 fraction
• ribosomes, tRNAs, tRNA synthetases, other soluble
  protein factors
• 20 amino acids
• GTP ATP
• Energy generating system
• to keep producing ATP and limit ADP
• PEP pyruvate kinase
• PEP ADP Pi ? pyruvate ATP
• Mg2 and K (NH4)

4
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).

5
How is right AUG selected for translation in
Prokaryotes?

• Many mRNAs contain a sequence preceding the start
  codon that base-pairs with the 3'-end of 16S rRNA
  (Shine-Dalgarno sequence)
• start
• 5'----GGAGG-------AUG-----3 mRNA
• 3'----CCUCC--------5' 16S rRNA
• Function helps position mRNA in ribosome.
• 2. The AUG itself is also very important
• 3. There is a S-D independent mode of translation
  initiation in E. coli
• 4. Translate internal ORFs of polycistronic mRNAs

S-D
6
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

7
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

8
Scanning (or Kozak) Model for Translation
Initiation in Eukaryotes
ATP
Fig. 17.16
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Apparent Exceptions to the Scanning Model?

• Translation of some mRNAs (5-10) doesnt 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
• C C R C C A U G G Rpurine
• Positions -3 and 4 are particularly important,
  based on mutagenesis studies

10
Fig. 17.18
Effect of the context of an upstream barrier
ATG on initiation of preproinsulin mRNA.
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.

11
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.
12
Conclusions

• An upstream AUG does not interfere if its
  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
  dont fall off the mRNA after translating such a
  short ORF.
• In natural mRNAs, upstream ORFs are very short,
  unless they have a regulatory role.

13
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
14
Effect of RNA Secondary Structure in the 5 UTR
(Leader)
Poorly translated
Translated well
Not translated
Trans. well
Adapted from Fig. 17.20
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Conclusions

• Secondary structure (hairpin) at very 5 end of
  RNA can prevent 40S subunit from binding
• Scanning ribosomes can melt out some hairpins (
  ?G -30 kcal/mole), but not highly stable ones (
  ?G -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).

16
Summary of translation initiation in Eukaryotes.
Resists binding to 60S subunit
Fig. 17.22
17
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

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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
• eIF4G - versatile adaptor
• eIF4A - RNA helicase
• eIF4E - binds the Cap

Fig. 17.25
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eIF4A and eIF4B

• eIF4A
• also exists outside of the eIF4F complex
• contains a DEAD motif (aspartate-glutamate-alanine
  -aspartate) 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

17.26
Role in translation Unwind hairpins in the 5
UTRs
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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?
21
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 cant bind eIF4E. eIF4G binds a
viral protein (X), that binds to the IRES,
promoting translation of the uncapped viral mRNAs.
17.27
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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

23
Toe-printing assay for determining where the
leading edge of a ribosome (or ribosomal subunit)
is on a mRNA
Fig. 17.31
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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.
eIF1eIF1A also convert Complex I into Complex II
when added after Complex I has formed (lane 8).
Fig. 17.32