A reassessment of the response of the bacterial ribosome to the frameshift stimulatory signal of the human immunodeficiency virus type 1.
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.
Léger
Mélissa
M
Département de Biochimie, Université de Montréal, 2900, boul. Edouard-Montpetit, D-353, Québec, H3T 1J4, Canada.
Sidani
Sacha
S
Brakier-Gingras
Léa
L
D018388:Codon, Terminator; D004926:Escherichia coli / Q000378:metabolism*; D017930:Genes, Reporter; D015497:HIV-1 / Q000378:metabolism*; D009154:Mutation; D009690:Nucleic Acid Conformation; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012367:RNA, Viral / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism*
D018388:Codon, Terminator; D012336:RNA, Ribosomal, 16S; D012367:RNA, Viral