Evidence that uncharged tRNA can inhibit a programmed translational frameshift in Escherichia coli.
In the modified release factor 2 (RF2) programmed translational frameshift (with a sense codon replacing the wild-type in-frame UGA codon at the shift site), ribosomes shift +1 into the reading frame for an out-of-frame reporter fused to the frameshift sequence. Partitioning of ribosomes between the out-of-frame shift and in-frame reading depends on the codon at the shift site and on the levels of tRNA decoding the in-frame codon. Overexpression of a tRNA species cognate to the in-frame codon at the shift site significantly reduces the frequency of frame-shifting, presumably by facilitating in-frame reading, which would reduce production of the out-of-frame reporter. However, since overexpression of a tRNA increases levels of both charged and uncharged tRNA, it is possible that uncharged cognate tRNA might be able to reduce the frequency of the frameshift, by entering the A site on the ribosome. To test this, we manipulated charged and uncharged tRNA levels in vivo, using the tryptophan analog tryptophan hydroxamate, which increases the proportion of uncharged tRNA(Trp) by competing with cognate amino acid tryptophan for tryptophanyl-tRNA synthetase, thereby reducing protein synthesis. We report here that a slight but reproducible reduction in the relative frequency of the frameshift is observed when tryptophan hydroxamate is added to cells containing the modified RF2 shift with UGG (Trp codon) at the shift site. When tRNA(Trp) is overexpressed from another plasmid, the shift frequency drops three- to fourfold, as expected, however, this reduction is still seen in the presence of the analog. Thus, under conditions when most of the tRNA(Trp) is apparently uncharged, excess tRNA(Trp) still causes a significant reduction in the frameshift when UGG is at the shift site, providing evidence that uncharged cognate tRNA also can inhibit this frameshift.
Gao
W
W
Department of Microbiology and Molecular Genetics, Graduate School of Biomedical Sciences, University of Medicine and Dentistry of New Jersey, Newark 07103, USA.
Jakubowski
H
H
Goldman
E
E
D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism; D003062:Codon / Q000235:genetics; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics*; D016368:Frameshift Mutation; D016296:Mutagenesis; D014176:Protein Biosynthesis / Q000235:genetics*; D012364:RNA, Transfer, Trp / Q000235:genetics* / Q000378:metabolism; D012270:Ribosomes / Q000235:genetics* / Q000378:metabolism; D014364:Tryptophan / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology
D003062:Codon; D012364:RNA, Transfer, Trp; C095117:tryptophanhydroxamate; D014364:Tryptophan; D000604:Amino Acyl-tRNA Synthetases