Transcription attenuation-mediated control of leu operon expression: influence of the number of Leu control codons.

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RESUMO

Four adjacent Leu codons within the leu leader RNA are critically important in transcription attenuation-mediated control of leu operon expression in Salmonella typhimurium and Escherichia coli (P. W. Carter, D. L. Weiss, H. L. Weith, and J. M. Calvo, J. Bacteriol. 162:943-949, 1985). The leader region from S. typhimurium was altered by site-directed mutagenesis to produce constructs having between one and seven adjacent Leu codons, all CUA. leu operon expression was measured in strains containing six of these constructs, each integrated into the chromosome in a single copy. Operon expression was sufficiently high that all strains grew in minimal medium unsupplemented by leucine. Expression of the operon was measured in strains cultured in such a way that their growth was limited by the intracellular concentration of either leucine or of leucyl-tRNA. In general, the leu operon for each construct responded similarly to the parent construct in terms of the degree of expression as a function of the degree of limitation. However, a strain containing (CUA)1 and, to a certain extent, a strain having (CUA)2 responded somewhat more sluggishly and strains containing (CUA)6 and (CUA)7 responded more sensitively to limitations than did the parent construct. In addition, DNA fragments containing the leu promoter and leader region were used as templates in in vitro transcription reactions employing purified RNA polymerase. With nucleoside triphosphate concentrations of 200 microM, RNA polymerase paused during transcription of the leu leader region at a site about 95 bp downstream from the site of transcription initiation. The halftimes of the pause were 1 min at 37 degrees C and 3 min at 22 degrees C. The pause was lengthened substantially when the GTP concentration was lowered to 20 micromoles. Our results are interpreted most easily in terms of an all-or-none model. Given two Leu control codons, the operon responds with nearly maximum output over a wide range of leucine limitation, and that outcome does not change much with increasing numbers of control codons.

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