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Mapping the cAMP receptor protein contact site on the α subunit of Escherichia coli RNA polymerase
Author(s) -
Zou Chao,
Fujita Nobuyuki,
Igarashi Kazuhiko,
Ishihama Akira
Publication year - 1992
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 - 10.1111/j.1365-2958.1992.tb01437.x
Subject(s) - biology , rna polymerase , camp receptor protein , microbiology and biotechnology , mutant , gene , transcription (linguistics) , site directed mutagenesis , escherichia coli , protein subunit , polymerase , genetics , gene expression , promoter , linguistics , philosophy
Summary The C ‐terminal region (amino acid residues 236–329) of the Escherichia coli RNA polymerase α subunit carries the contact site I for positive transcription factors. For detailed mapping of the contact site for the cAMP receptor protein (CRP), we made a library of mutant rpoA by polymerase chain reaction (PCR) mutagenesis, such that each should carry a single mutation on average and exclusively in the C ‐terminal half of the rpoA gene, and then screened this library for mutants with decreased expression of the lacZ gene. Reconstituted holoenzyme containing the mutant a subunits transcribed galP1 but not lacP1 in vitro in the presence of cAMP–CRP. DNA sequence determination of several ‘Lac ‐ ’ mutant rpoA genes revealed that all had mutations clustered within a short segment near the C ‐terminus of α between amino acid residues 265 and 270. A cluster of contact sites appear to exist within the contact site I region, each comprising of about five amino acids and responding in molecular communication with a different transcription factor(s).