Analysis of the roles of tRNA structure, ribosomal protein L9, and the bacteriophage T4 gene 60 bypassing signals during ribosome slippage on mRNA

A 50-nucleotide coding gap divides bacteriophage T4 gene 60 into two open r eading frames. In response to cis-acting stimulatory signals encrypted in t he mRNA, the anticodon of the ribosome-bound peptidyl tRNA dissociates from a GGA codon at the end of the first open reading frame and pairs with a GG A codon 47 nucleotides downstream just before the second open reading frame . Mutations affecting ribosomal protein L9 or tRNA(2)(Gly), the tRNA that d ecodes GGA, alter the efficiency of bypassing. To understand the mechanism of ribosome slippage, this work analyzes the influence of these bypassing s ignals and mutant translational components on -1 frameshifting at (G) under bar (GG) under barA and hopping over a stop codon immediately flanked by t wo GGA glycine codons (stop-hopping). Mutant variants of tRNA(2)(Gly) that impair bypassing mediate stop-hopping with unexpected landing specificities , suggesting that these variants are defective in ribosomal P-site codon-an ticodon pairing. In a direct competition between -1 frameshifting and stop- hopping, the absence of L9 promotes stop-hopping at the expense of -1 frame shifting without substantially impairing the ability of mutant tRNA(2)(Gly) variants to re-pair with the mRNA by sub-optimal pairing. These observatio ns suggest that L9 defects may stimulate ribosome slippage by enhancing mRN A movement through the ribosome rather than by inducing an extended pause i n translation or by destabilizing P-site pairing. Two of the bypassing signals, a cis-acting nascent peptide encoded by the f irst open reading frame and a stemloop signal located in the 5' portion of the coding gap, stimulate peptidyl-tRNA slippage independently of the rest of the gene 60 context. Evidence is presented suggesting that the nascent p eptide signal may stimulate bypassing by destabilizing P-site pairing. (C) 2001 Academic Press.