Interaction of a putative repressor protein with an extended control region of the Bacillus subtilis pur operon.
Transcription of the Bacillus subtilis pur operon is regulated independently by adenine and guanine levels in the growth medium. Transcription initiation is regulated by adenine, transcription attenuation by guanine (Ebbole, D. J., and Zalkin, H. (1987) J. Biol. Chem. 262, 8274-8287). A purC-lacZ fusion was constructed to monitor gene expression. Expression was reduced 11- and 18-fold by excess adenine and guanine, respectively. We have characterized the mechanism for the adenine-mediated control of transcription initiation. Deletions of DNA between nucleotides -193 and -37, relative to the start of transcription, resulted in high level constitutive expression and thus identified a control site for negative regulation. A candidate for a pur operon repressor protein was identified in a cell extract and was partially purified. This protein interacts specifically with an extended 5' flanking region of the pur operon that includes the control site identified by deletion analysis. DNase I footprinting defined the region of DNA-protein interaction between nucleotides -145 and -29. The protein-DNA interaction was not dependent on adenine, adenosine, or adenine nucleotides in vitro. These results suggest that a complex interaction of protein with an extended control region may repress transcription by blocking the -35 promoter site. This protein-DNA interaction is unlike that which regulates pur gene expression in Escherichia coli.
Ebbole
D J
DJ
Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907.
Zalkin
H
H
D000225:Adenine / Q000494:pharmacology; D001412:Bacillus subtilis / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites; D002852:Chromatography, Ion Exchange; D004250:DNA Topoisomerases, Type II / Q000378:metabolism; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism*; D003850:Deoxyribonuclease I / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005786:Gene Expression Regulation / Q000187:drug effects; D006147:Guanine / Q000494:pharmacology; D008969:Molecular Sequence Data; D009876:Operon; D011401:Promoter Regions, Genetic; D011685:Purine Nucleotides / Q000235:genetics*; D012097:Repressor Proteins / Q000302:isolation & purification / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects
D004269:DNA, Bacterial; D011685:Purine Nucleotides; D012097:Repressor Proteins; D014157:Transcription Factors; D006147:Guanine; D003850:Deoxyribonuclease I; D004250:DNA Topoisomerases, Type II; D000225:Adenine