Increased ribosomal accuracy increases a programmed translational frameshift in Escherichia coli.
We have tested the effect of increased ribosomal fidelity on a modified version of the programmed release factor 2 (RF2) translational frameshift. In the constructs tested, the original UGA codon at the site of the shift was replaced by either of two sense codons, UGG (tryptophan), which allows a frameshift of approximately 13%, or CUG (leucine), which allows a frameshift of only approximately 2%. We confirmed the results of Curran and Yarus [Curran, J. F. & Yarus, M. (1989) J. Mol. Biol. 209, 65-77] in a wild-type ribosomal host, including a reduction of the UGG shift following induction of tRNA(Trp) from a plasmid copy of the tRNA gene. But to our surprise, in a hyperaccurate streptomycin pseudo-dependent host, the UGG frameshift increased to more than 50%. When we added a tRNA(Trp) plasmid to these cells, induction of the tRNA(Trp) gene reduced the shift back to approximately 7%. Messenger RNA levels did not vary greatly under these different induced conditions. Other increased accuracy alleles also showed increased frameshifting with UGG at the frameshift site. All increased accuracy alleles led to slower translation rates, and there appeared to be a proportionality between the extent of reduction of synthesis for the in-frame reporter and the extent of UGG frameshift for the out-of-frame reporter. There were little effects of increased accuracy on the lower level CUG frameshift. However, over-production of the cognate tRNA(1Leu) dramatically reduced even this lower level of shift, despite the fact that tRNA(1Leu) is already the most abundant isoacceptor in Escherichia coli. These results can be rationalized by following the hypothesis of Curran and Yarus as follows: with wild-type ribosomes, limited availability of tRNA(Trp) (about 1% of total tRNA) facilitates a pause at the UGG codon (due to the vacant A site), allowing increased opportunity for ribosome realignment. Excess tRNA(Trp) reduces the time the A site is vacant and thus reduces the frameshift. The slower hyperaccurate ribosomes increase the pause time and thus increase the opportunity for shifting, a process again reversed by increasing the in-frame cognate tRNA(Trp). These data provide strong support for a model in which the extent of ribosome pause time at a programmed frameshift site is a major determinant in the efficiency of the frameshift and in which tRNA availability can be a major influence on this process.
Sipley
J
J
Department of Microbiology and Molecular Genetics, New Jersey Medical School, Newark.
Goldman
E
E
D001483:Base Sequence; D003062:Codon / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D016368:Frameshift Mutation; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012343:RNA, Transfer / Q000235:genetics / Q000378:metabolism*; D012356:RNA, Transfer, Leu / Q000235:genetics / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism*; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D003062:Codon; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D012356:RNA, Transfer, Leu; D012343:RNA, Transfer; D001616:beta-Galactosidase