GENE EXPRESSION AND ORGANIZATION 
Raymond F. Gesteland, Ph.D, Investigator 
I. Unusual Translation Events. 
A. Frameshifting and hopping (Dr. John F. Atkins, 
Michael O'Connor, Therese Tuohy, and Norma 
Wills). Michael O'Connor completed work on mu- 
tants of tRNA^^' that enhance short-distance hop- 
ping from one valine codon to another. One class 
of mutants has a single-base insertion in its antico- 
dons. The hopping efficiency of these mutants 
dropped sharply with increasing distance. Low-effi- 
ciency hopping occurs over three tandem stop co- 
dons, whereas in certain mRNA contexts, hopping 
between two valine codons spaced two nucleotides 
apart occurs with 20% efficiency. The other class of 
mutants has a base substitution in the third nucleo- 
tide from the 3' end of the tRNA, changing CCA to 
GCA or ACA. Norma Wills, who is investigating the 
tRNA from these mutants, found (in collaboration 
with Dr. Lionello Bossi) an equivalent change in a 
mutant tRNA"'* that altered the regulation of the 
histidine biosynthetic operon. This class of mutants 
may provide information about the interaction be- 
tween the 3' end of tRNA and the peptidyl transfer- 
ase region of ribosomal RNA. 
B. Large hops (Dr. Robert Weiss and Diane Dunn). 
The laboratory is continuing to examine the hop- 
ping of ribosomes over a 50 nucleotide (nt) inter- 
ruption in the mRNA of bacteriophage T4 gene 60 
with nearly 100% efficiency. A mutational analysis of 
the mRNA elements required has unexpectedly re- 
vealed that the peptide sequence encoded in the 
138 nt 5' of the jump site is required for high-level 
bypass of the interruption, as if the growing poly- 
peptide chain itself is somehow promoting this 
event. Mutations that disrupt the interrupt also in- 
dicate that the gene 60 jump is analogous to tRNA 
hopping, previously observed at low levels in Es- 
cherichia coli. Also it is apparent that appropriate 
secondary structure is needed at certain sequences 
of the interrupt. 
C. Retroviral frameshifts (Dr. R. Weiss and D. 
Dunn). With most retroviruses the coupling oigag- 
pol expression is effected by ribosomal frameshift- 
ing; the sequences required include certain tandem 
codons plus some downstream sequence that at 
least in one case involves a pseudoknot structure. 
Expression of retroviral ribosomal frameshift sites 
in E. coli as retroviral gag-pol/lacZ fusions has 
shown that these prokaryotic ribosomes can re- 
spond to the retroviral mRNA signals that provoke 
high-level ribosomal frameshifting. This has pro- 
vided a convenient system in which to analyze the 
components of these frameshift sites comprising a 
special slippery tRNA context and a 3 -flanking 
mRNA element. 
D. Plant viruses (Dr. James Skuzeski and Linda 
Nichols). Several plant RNA viruses encode read- 
through proteins, where suppressor tRNA reading 
of a "leaky" stop codon has been implicated. How- 
ever, in three unrelated viruses, the sequence 
around the read-through site is conserved (CAA- 
TAG-CAA-TTA), with an amber stop codon flanked 
by two glutamine codons. The recent demonstra- 
tion that tRNAs can hop over stops flanked by ho- 
mologous codons in E. coli suggests an alternative 
explanation. Hopping is being tested by transfec- 
tion of tobacco protoplasts with constructs contain- 
ing a (3-glucuronidase reporter gene. Identity of the 
3' CAA codon is crucial as predicted if hopping 
were involved. Surprisingly, however, read-through 
levels are also drastically lowered by several muta- 
tions in the UUA leucine codon, and it is unclear 
how this fits with either the suppression model or 
hopping. Protein sequence information is crucial. 
Stably transformed tobacco cell lines that express 
the gene have been established for this purpose. 
E. T7 frameshifting (Barry Condron). The major 
capsid gene of bacteriophage T7 has a frameshift 
product. Experiments with fusions suggest that se- 
quences far removed on the mRNA may be in- 
volved. 
F. Messenger RNA stability (Lori Wagner and Dr. 
Weiss). Sequence elements near the 5' end of mRNAs 
can affect mRNA stability. It is unknown if these act 
by determining nuclease sensitivity directly or by al- 
tering ribosome initiation frequency. A single-base 
change in the fourth codon of lacZ decreases 
mRNA stability threefold. The effects of translation 
initiation on mRNA stability are being tested by in- 
serting oligonucleotides into the lacZ gene, which 
alters the wild-type Shine-Dalgarno (SD) sequence 
and the initiation codon. The less efficient SDs cor- 
relate with message instability. The results may be 
explained by a 30 S subunit binding the SD region 
and protecting a ribonuclease-sensitive region. 
Continued 
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