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Département de Biochimie, Université de Montréal, Montréal, Québec, H3T 1J4, Canada
Reprint requests to: Léa Brakier-Gingras, Département de Biochimie, Université de Montréal, 2900, boul. Édouard-Montpetit, D-353, Montréal, Québec, H3T 1J4, Canada; e-mail: lea.brakier.gingras{at}umontreal.ca; fax: (514) 343-2210.
HIV-1 uses a programmed -1 ribosomal frameshift to produce the precursor of its enzymes. This frameshift occurs at a specific slippery sequence followed by a stimulatory signal, which was recently shown to be a two-stem helix, for which a three-purine bulge separates the upper and lower stems. In the present study, we investigated the response of the bacterial ribosome to this signal, using a translation system specialized for the expression of a firefly luciferase reporter. The HIV-1 frameshift region was inserted at the beginning of the coding sequence of the luciferase gene, such that its expression requires a –1 frameshift. Mutations that disrupt the upper or the lower stem of the frameshift stimulatory signal or replace the purine bulge with pyrimidines decreased the frameshift efficiency, whereas compensatory mutations that re-form both stems restored the frame-shift efficiency to near wild-type level. These mutations had the same effect in a eukaryotic translation system, which shows that the bacterial ribosome responds like the eukaryote ribosome to the HIV-1 frameshift stimulatory signal. Also, we observed, in contrast to a previous report, that a stop codon immediately 3' to the slippery sequence does not decrease the frameshift efficiency, ruling out a proposal that the frameshift involves the deacylated-tRNA and the peptidyl-tRNA in the E and P sites of the ribosome, rather than the peptidyl-tRNA and the aminoacyl-tRNA in the P and A sites, as commonly assumed. Finally, mutations in 16S ribosomal RNA that facilitate the accommodation of the incoming aminoacyl-tRNA in the A site decreased the frameshift efficiency, which supports a previous suggestion that the frameshift occurs when the aminoacyl-tRNA occupies the A/T entry site.
Keywords: bacterial ribosome; HIV-1 programmed –1 ribosomal frameshift; ribosomal RNA mutagenesis
Abbreviations: aa-tRNA, aminoacyl-tRNA; EF-Tu, elongation factor Tu; HIV-1, human immunodeficiency virus type 1; IPTG, isopropyl-ß-D-thiogalactopyranoside; p-tRNA, peptidyl-tRNA; rRNA, ribosomal RNA
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