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cis ‐Acting regulatory sequences for antitermination in the transcript of the Bacillus subtilis hut operon and histidine‐dependent binding of HutP to the transcript containing the regulatory sequences
Author(s) -
Oda Masanao,
Kobayashi Nobuhiro,
Ito Akiko,
Kurusu Yasurou,
Taira Kazunari
Publication year - 2000
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1046/j.1365-2958.2000.01795.x
Subject(s) - antitermination , biology , operon , histidine , terminator (solar) , electrophoretic mobility shift assay , bacillus subtilis , rna , random hexamer , microbiology and biotechnology , transcription (linguistics) , binding site , protein secondary structure , biochemistry , gene , genetics , gene expression , escherichia coli , amino acid , ionosphere , linguistics , philosophy , physics , astronomy , bacteria
The location of the cis ‐acting regulatory region for histidine‐dependent antitermination of the Bacillus subtilis hut operon was determined. A secondary structure, whose sequences partially overlap with the downstream terminator, was found in the regulatory region of the hut transcript. Mutational analysis of the regulatory region showed that the secondary structure was required for histidine‐dependent antitermination. An electrophoretic mobility‐shift assay demonstrated that, in response to the presence of histidine and Mg 2+ , purified HutP bound hut RNA bearing putative secondary structure but not RNA lacking the potential to form putative secondary structure. Native gel electrophoresis showed that HutP existed as a hexamer. A filter‐binding assay revealed that the concentration of histidine required for half‐maximal binding of HutP to RNA was 3.1 mM and that the K d for binding of HutP to RNA was ≈ 0.56 µM in the presence of histidine. These results suggested that putative secondary structure in the regulatory region of hut mRNA could function as an antiterminator to inhibit the formation of the terminator structure and that HutP causes expression of the hut structural genes by binding to the putative antiterminator structure in response to the presence of histidine.