Ribosome bypassing elicited by tRNA depletion.
Ribosome bypassing refers to the ability of the ribosome::peptidyl-tRNA complex to slide down the message without translation to a site several or dozens of nucleotides downstream and resume protein chain elongation there. The product is an isoform of a protein with a 'coding' gap corresponding to the region of the message which was bypassed. Previous work showed that ribosome bypassing was strongly stimulated at 'hungry' codons calling for a tRNA whose aminoacylation was limited. We have now used the 'minigene' phenomenon to ascertain whether depletion of the pool of specific isoacceptors has a similar effect. High level expression of plasmid-borne minigenes results in the sequestration as peptidyl-tRNA of tRNA cognate to the last triplet of the minigene, thereby limiting protein synthesis for lack of the tRNA in question. We find that induction of a minigene ending in AUA stimulates bypassing at an AUA codon, but not in a control sequence with AGA at the test position; induction of a minigene ending in AGA stimulates bypassing at the latter but not the former. Induction of the AUA minigene also stimulates both leftward and rightward frameshifting at 'shifty' sequences containing an AUA codon. The normal, background frequency of bypassing at an AUA codon is markedly reduced by increasing the cellular level of the tRNA which reads the codon. Thus, the frequency of bypassing can be increased or decreased by lowering or raising the concentration of a relevant tRNA isoacceptor. These observations suggest that the occurrence of ribosome bypassing reflects the length of the pause at a given codon.
Lindsley
Dale
D
University of Washington, Department of Genome Sciences, Box no. 357730, Seattle 98105, USA.
Gallant
Jonathan
J
Guarneros
Gabriel
G
D000595:Amino Acid Sequence; D001483:Base Sequence; D003062:Codon; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D018965:Frameshifting, Ribosomal; D005796:Genes / Q000235:genetics; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D012343:RNA, Transfer / Q000378:metabolism*; D012270:Ribosomes / Q000235:genetics*
D003062:Codon; D012343:RNA, Transfer