Identification of a cis-acting regulatory sequence responsible for the repression of brnQ in Salmonella typhimurium

• Published in: Biochimica et biophysica acta 1999-05, Vol.1445 (2), p.196-206
• Main Authors: Ohnishi, Kuniharu, Matsubara, Keiko, Hattori, Yoshihiko, Sadanari, Hidetaka, Yamada, Rie, Fukuda, Shizuo
• Format: Article
• Language: English
• Subjects: Artificial Gene Fusion; Base Sequence; Branched-chain amino acid; Gene Expression Regulation; Genes, Bacterial; Lrp; Molecular Sequence Data; Plasmids; Regulatory Sequences, Nucleic Acid; Repressor; Repressor Proteins - genetics; Salmonella typhimurium; Salmonella typhimurium - genetics; Transport
• ISSN: 0167-4781; 0006-3002; 1879-2634
• DOI: 10.1016/S0167-4781(99)00043-3

brnQ is the gene encoding the LIV-II transport system for branched-chain amino acids in Salmonella typhimurium. The expression of the gene is transcriptionally repressed by an excess of glycyl- l-leucine added to the bacterial culture. To investigate the mechanism of regulation, we constructed brnQ-lacZ translational fusions with various deletions upstream from the promoter of brnQ, and examined the effects of the deletions on the regulation. We found a cis-acting region, 5′-GTGTTTTA-3′, for the repression of brnQ expression, which was located 94 base pairs upstream from the transcription start site. Removal of the sequence resulted in derepression of brnQ. Two homologous sequences were found 45 base pairs downstream and 42 base pairs upstream from the sequence. We designated these sequences as O1, O2, and O3, in the order from the sequence proximal to the promoter to that distal to the promoter, respectively. The gleR1 mutation, which we reported previously to be a regulatory mutation enhancing transcription of brnQ, was a G-to-T transversion in the O1 sequence 50 base pairs upstream from the transcription start site. Insertion of five nucleotides between O1 and O2 resulted in derepression of brnQ. Further insertion of five nucleotides did not restore the original regulation of brnQ, indicating the importance of the proper spacing of these sequences. We also showed that the protein product of livS, the gene responsible for regulation of the LIV-I transport system, may bind to the O2 sequence. Furthermore, LivS was shown to be an allele of Lrp based on complementation experiments.