Effects of tRNA(1Leu) overproduction in Escherichia coli.
Strains of Escherichia coli have been produced which express very high levels of the tRNA(1Leu) isoacceptor. This was accomplished by transforming cells with plasmids containing the leuV operon which encodes three copies of the tRNA(1Leu) gene. Most transformants grew very slowly and exhibited a 15-fold increase in cellular concentrations of tRNA(1Leu). As a result, total cellular tRNA concentration was approximately doubled and 56% of the total was tRNA(1Leu). We examined a number of parameters which might be expected to be affected by imbalances in tRNA concentration: in vivo tRNA charging levels, misreading, ribosome step time, and tRNA modification. Surprisingly, no increase in intracellular ppGpp levels was detected even though only about 40% of total leucyl tRNA was found to be charged in vivo. Gross ribosomal misreading was not detected, and it was shown that ribosomal step times were reduced between two- and threefold. Analyses of leucyl tRNA isolated from these slow-growing strains showed that at least 90% of the detectable tRNA(1Leu) was hypomodified as judged by altered mobility on RPC-5 reverse-phase columns, and by specific modification assays using tRNA(m1G)-methyltransferase and pseudo-uridylate synthetase. Analysis of fast-growing revertants demonstrated that tRNA concentration per se may not explain growth inhibition because selected revertants which grew at wild-type growth rates displayed levels of tRNA comparable to that of control strains bearing the leuV operon. A synthetic tRNA(1Leu) operon under the control of the T7 promoter was prepared which, when induced, produced six- to sevenfold increases in tRNA(1Leu) levels. This level of tRNA(1Leu) titrated the modification system as judged by RPC-5 column chromatography. Overall, our results suggest that hypomodified tRNA may explain, in part, the observed effects on growth, and that the protein-synthesizing system can tolerate an enormous increase in the concentration of a single tRNA.
Wahab
S Z
SZ
Department of Microbiology, Medical College of Virginia, Virginia Commonwealth University, Richmond 23298-0678.
Rowley
K O
KO
Holmes
W M
WM
D001426:Bacterial Proteins / Q000096:biosynthesis; D001483:Base Sequence; D004269:DNA, Bacterial / Q000235:genetics; D004274:DNA, Recombinant / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D005814:Genes, Viral; D006159:Guanosine Tetraphosphate / Q000032:analysis; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009876:Operon; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D012323:RNA Processing, Post-Transcriptional; D012329:RNA, Bacterial / Q000096:biosynthesis*; D012356:RNA, Transfer, Leu / Q000096:biosynthesis*; D012270:Ribosomes / Q000378:metabolism; D013604:T-Phages / Q000235:genetics
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004274:DNA, Recombinant; D012329:RNA, Bacterial; D012356:RNA, Transfer, Leu; D006159:Guanosine Tetraphosphate