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research-article

Premature termination of in vivo transcription of a gene encoding a branched-chain amino acid transport protein in Escherichia coli.

Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor 48109-0606.

ABSTRACT

Previous studies have suggested that control of expression of genes of the LIV-I permease system for the high-affinity transport of branched-chain amino acids in Escherichia coli involves modulation in the frequency of mRNA elongation. Mutation of the Rho transcription termination factor and shortages of charged leucyl-tRNA have been shown to alter LIV-I transport activity. Rho-dependent transcription termination regulated by shortages of charged leucyl-tRNA at sites preceding structural genes has been proposed to account for their role in regulation of LIV-I transport. Transcription of the livJ-binding protein gene, encoding one of the periplasmic components of the LIV-I system, was analyzed in vivo with strains which lack repression of the LIV-I genes and harbor a temperature-sensitive allele for either leucyl-tRNA synthetase or Rho factor. Analysis of mRNA synthesis by DNA-RNA hybridization in the various mutant strains indicated that both shortages of leucyl-tRNA caused by inactivation of the temperature-sensitive leucyl-tRNA synthetase and inactivation of the Rho factor were associated with increased synthesis of livJ mRNA. Nuclease protection and gel electrophoresis studies detected prematurely terminated transcripts corresponding in size to the leader region of livJ mRNA. Accumulations of these short transcripts were suppressed in strains harboring temperature-sensitive alleles for either leucyl-tRNA synthetase or Rho factor. These results provide support for the hypothesis that expression of livJ involves Rho-dependent transcription termination in which antitermination is associated with the intracellular availability of aminoacyl leucyl-tRNA.