PROKARYOTIC RIBOSOMES RECODE THE HIV-1 GAG-POL-1 FRAMESHIFT SEQUENCE BY AN E P SITE POST-TRANSLOCATION SIMULTANEOUS SLIPPAGE MECHANISM/

The mechanism favoured for -1 frameshifting at typical retroviral site s is a pre-translocation simultaneous slippage model. An alternative p ost-translocation mechanism would also generate the same protein seque nce across the frameshift site and therefore in this study the strateg ic placement of a stop codon has been used to distinguish between the two mechanisms. A 26 base pair frameshift sequence from the HIV-1 gag- pol overlap has been modified to include a stop codon immediately 3' t o the heptanucleotide frameshift signal, where it often occurs natural ly in retroviral recoding sites. Stop codons at the 3'-end of the hept anucleotide sequence decreased the frameshifting efficiency on prokary ote ribosomes and the recoding event was further depressed when the le vels of the release factors in vivo were increased, in the presence of elevated levels of a defective release factor 2, frameshifting effici ency in vivo was increased in the constructs containing the stop codon s recognized specifically by that release factor. These results are co nsistent with the last six nucleotides of the heptanucleotide slippery sequence occupying the ribosomal E and P sites, rather than the P and A sites, with the next codon occupying the A site and therefore with a post-translocation rather than a pre-translocation -1 slippage model .