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Program
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Mentor
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Research
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| The faithful
production of proteins requires a series of molecular
processes that each must be performed accurately. Occasionally
the translational machinery makes spontaneous errors,
some of the most damaging of which are shifts in the reading
frame. These frameshifts result in truncated proteins,
which are more often than not biologically inactive. Certain
mRNAs are able to preferentially induce frameshifting
at frequencies much higher than those found for spontaneous
frameshifts. Some of these messages contain cis acting
sequences that can alter the inherent accuracy of translation
and produce a shift out of the normal reading frame, while
others rely only on non-canonical tRNA-ribosome interactions.
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| One of these
sequences is found in the Ty3 element in yeast. The Ty3
element produces a gag analog of GAG3 in the 5' reading
frame and expresses a pol analog POL3 in the 3' reading
frame, but produces the GAG-POL fusion product via a +1
frameshift event (Farabaugh et al., 1993). The sequence
of the frameshift site in the Ty3 element is GCG AGU U
with the frameshifting event occurring due to the out
of frame binding of the aminoacyl-tRNA to GUU in the A
site of the ribosome. An unusual tRNA interaction in the
P site (Figure 1) apparently causes a reduction
in the affinity for the cognate tRNA in the A site. |
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Figure
1. Ty3 frameshift site showing the clash in the
wobble position when tRNA(Ala, IGC) is in the P
site |
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Figure
2. Slow recognition of the A site in frame by tRNA(Ser,GCU)
aids in the acceptance of the out of frame tRNA(Val,IAC). |
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| Potentially,
a mismatch in the wobble position may induce frameshifting
(Stahl et. al., 2001). This is aided by the fact that
the in frame codon is recognized slowly while the codon
that binds out of frame is recognized much more rapidly
(Figure 2) (Farabaugh, 1996). Frameshift errors
are increased in the Ty3 element by a short 14 nucleotide
sequence downstream of the frameshift site that is called
the stimulator. The Ty3 stimulator increases frameshifting
up to 7.5 fold. The spacing between the stimulator and
the frameshift site is important since as little as a
1 nucleotide change can eliminate this effect (Li, 2000).
The stimulator is complementary to part of the sequence
of Helix 18 in the rRNA (Figure 3), a region which
is important in maintaining translational accuracy. Helix
18 is thought to play an active role in the discrimination
between cognate and non-cognate interactions in the A
site (Powers and Noller, 1991). |
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The
model for the stimulator effect that has developed
in our lab is that base pairing between the context
and Helix 18 may inhibit the ability of Helix 18
to form a structure necessary for accurate selection
of a tRNA in the A site. This decreases the affinity
for cognate tRNA. This effect, combined with the
aberrant P site interaction, and slow recognition
AGU codon in the A-site all increase the likelihood
of the acceptance of the out of frame tRNA. |
Figure
3.A. Putative Helix 18- stimulator
interaction |
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| The stimulator
also has an effect on initiation accuracy in E. coli.
Preliminary data has shown that the stimulator, when placed
downstream of the initiation site, was able to increase
initiation at UUG up to 7 fold. This data suggests that
Helix 18 also may play a role in initiation accuracy.
How then does an element that effects tRNA selection in
the A-site effect the fidelity of initiation, which is
a P site event? Recent structural work on the ribosome-Initiation
Factor 1 (IF1) interaction may shed some light on this
problem. Binding of IF1 into the A site induces conformational
changes in the ribosome and it has been suggested that
these alterations may be transmitted to the accuracy center
of the ribosome, resulting in structural changes in the
P site which would allow for proper selection of initiator
tRNA (Dahlquist, 2001). Carter et. al. (2001) recently
solved the crystal structure of IF1 bound to the 30S subunit,
supporting previous data from chemical probing experiments
of IF1 bound to the 30S subunit by showing an interaction
between Helix 18 and IF1. Dahlquist and Puglisi (2001)
have suggested that these interactions between Helix 18
and IF1 are not essential for binding but may perform
some other enzymatic function. It is possible that the
stimulator, when immediately downstream of the initiation
codon interferes with the Helix 18-IF1 interaction, therefore
not allowing for the proper structure to be formed in
the P-site, resulting in inaccuracies during initiation. |
| References |
| Carter,
A. P., Clemons, W.M. Jr., Brodersen, D.E., Morgan-Warren,
R.J., Hartsch, T., Wimberly, B.T.,, and Ramakrishnan,
V. |
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Crystal
structure of an initiation factor bound to the 30S
ribosomal subunit..Science (2001) 291:498-501. |
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| Dahlquist,
K. D. and Puglisi, J. D. |
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Interaction of Translation Initiation Factor IF1
with the E. coli Ribosomal A site. J. Mol. Biol.
(2001) 299:1-15. |
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| Farabaugh,
P. |
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Programmed
Alternative Reading of the Genetic Code (Austin,
Texas, R. G. Landes Company). (1996). |
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| Farabaugh,
P. J., Zhao, H., and Vimaladithan, A. |
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A
novel programed frameshift expresses the POL3 gene
of retrotransposon Ty3 of yeast: frameshifting without
tRNA slippage. Cell (1993) 74:93-103. |
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| Li,
Z., Stahl, G. and Farabaugh P.J. |
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Programmed
+1 frameshifting stimulated by complementarity between
a downstream mRNA sequence and an error correcting
region of rRNA. RNA. (2000) 7:275-284. |
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| Powers,
T., and Noller, H. F. |
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A
functional pseudoknot in 16S ribosomal RNA. EMBO
J. (1991). 10:2203-2214. |
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Publications
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| Stahl,
G., McCarty, G. P.
and Farabaugh,
P. J. |
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Ribosome
structure: revisiting the connection between translational
accuracy and unconventional decoding. Trends
Biochem. Sci. (2002) 27:178-183. |
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| Stahl,
G., Ben Salem, S., Li, Z., McCarty,
G., Raman, A., Shah, M and Farabaugh,
P. J. |
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Programmed
+1 translational frameshifting in the yeast Saccharomyces
cerevisiae results from disruption of translational
error correction. Cold Spring Harb. Symp. Quant.
Biol. (2001) 66, in press |
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