Analysis of CRP-CytR interactions at the Escherichia coli udp promoter.
Multiprotein complexes regulate the transcription of certain bacterial genes in a sensitive, physiologically responsive manner. In particular, the transcription of genes needed for utilization of nucleosides in Escherichia coli is regulated by a repressor protein, CytR, in concert with the cyclic AMP (cAMP) activated form of cAMP receptor protein (CRP). We studied this regulation by selecting and characterizing spontaneous constitutive mutations in the promoter of the udp (uridine phosphorylase) gene, one of the genes most strongly regulated by CytR. We found deletions, duplications, and point mutations that affect key regulatory sites in the udp promoter, insertion sequence element insertions that activated cryptic internal promoters or provided new promoters, and large duplications that may have increased expression by udp gene amplification. Unusual duplications and deletions that resulted in constitutive udp expression that depended on the presence of CytR were also found. Our results support the model in which repression normally involves the binding of CytR to cAMP-CRP to form a complex which binds to specific sites in the udp promoter, without direct interaction between CytR protein and a specific operator DNA sequence, and in which induction by specific inducer cytidine involves dissociation of CytR from cAMP-CRP and the RNA polymerase interaction with cAMP-CRP bound to a site upstream of then transcription start point. The stimulation of udp expression by CytR in certain mutants may reflect its stabilization of cAMP-CRP binding to target DNA and illustrates that only modest evolutionary changes could allow particular multiprotein complexes to serve as either repressors or transcriptional activators.
Brikun
I
I
Department of Molecular Microbiology, Washington University Medical School, St. Louis, Missouri 63110, USA.
Suziedelis
K
K
Stemmann
O
O
Zhong
R
R
Alikhanian
L
L
Linkova
E
E
Mironov
A
A
Berg
D E
DE
D001426:Bacterial Proteins / Q000378:metabolism*; D001483:Base Sequence; D002352:Carrier Proteins; D002373:Cyclic AMP Receptor Protein / Q000378:metabolism*; D018983:DNA Footprinting; D004794:Enzyme Repression; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012097:Repressor Proteins / Q000378:metabolism*; D014158:Transcription, Genetic; D014530:Uridine Diphosphate / Q000032:analysis; D014543:Uridine Phosphorylase / Q000235:genetics*
D001426:Bacterial Proteins; D002352:Carrier Proteins; D002373:Cyclic AMP Receptor Protein; C048425:CytR protein, E coli; D029968:Escherichia coli Proteins; D012097:Repressor Proteins; D014530:Uridine Diphosphate; D014543:Uridine Phosphorylase