Nucleic Acids Research, Vol 27, Issue 24 4783-4791, Copyright © 1999 by Oxford University Press
MN Brunelle, C Payant, G Lemay and L Brakier-Gingras
A-1 frameshift event is required for expression of the pol gene when
ribosomes translate the mRNA of human immunodeficiency virus type-1
(HIV-1). In this study, we inserted the frameshift region of HIV-1 (a
slippery heptanucleotide motif followed by a stem-loop) in a reporter gene
coding for firefly luciferase. The ability of the corresponding mRNA,
generated by in vitro transcription, to be translated in an Escherichia
coli cell-free extract is the first demonstration that the HIV-1 frameshift
can be reproduced in a bacterial cell-free extract, providing a powerful
approach for analysis of the frameshift mechanism. The responses of the
frameshift signal to chloramphenicol, an inhibitor of peptide bond
formation, and spectinomycin, an inhibitor of translocation, suggest that
the frameshift complies with the same rules found in eukaryotic translation
systems. Furthermore, when translation was performed in the presence of
streptomycin and neamine, two error- inducing antibiotics, or with
hyperaccurate ribosomes mutated in S12, the frameshift efficiency was
increased or decreased, respectively, but only in the presence of the
stem-loop, suggesting that the stem-loop can influence the frameshift
through a functional interaction with the ribosomes.
ARTICLES
Expression of the human immunodeficiency virus frameshift signal in a bacterial cell-free system: influence of an interaction between the ribosome and a stem-loop structure downstream from the slippery site
Departement de Biochimie, Universite de Montreal, Montreal, Quebec H3T 1J4, Canada.
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