We propose to examine CytR, a most unusual transcriptional repressor in E. coli, a repressor that may block expression of its target genes without actually making base-specific contacts with DNA.The repression of genes under strong CytR control (nucleoside utilization; e.g. udp, cdd, deo) depends on cyclic AMP-activated cAMP receptor protein (CRP) binding to two sites separated by about 52 bp in target promoter regions. Although CytR exhibits considerable homology to classical (e.g. lacI) repressor proteins, and protects sequences between CRP sites from nuclease digestion in footprinting experiments, no obvious extended consensus emerges from the comparison of sequences between the CRP sites. Thus, in the extreme, CytR may simply bind to CRPs that, in turn, are bound to DNA with an approximately 52 bp spacing. The following five groups of experiments will be done to test this model and more generally elucidate how CytR acts: 1. CytR is autoregulatory and expressed at low levels. We will PCR-clone a promoterless cytR gene downstream of a tightly regulated lac promoter, so that levels of CytR protein can be varied from <10 to >1000 molecules per cell, using the IPTG lac inducer. Fine control of CytR levels will allow subtle effects of mutations in target promoter regions and in CytR itself to be detected with sensitivity. 2. We will sequence Mironov's mutant alleles of the udp promoter region, using efficient PCR and linear amplification methods. 3. We will use combinatorial in vitro mutagenesis to further identify features in udp and related promoter regions that are important for repression by CytR. 4. We will select and characterize mutant cytR alleles with altered repression specificity, able to act on constitutive mutant promoter alleles, or with CytR-resistant crp alleles. 5. We will examine CytR-target site interactions using chemical probe methods. These experiments will be collaborative between the groups of Berg (Washington University, St. Louis) and Mironov (Inst. Genetics Selection Industrial Microorganisms, Moscow), and are expected to have numerous benefits for each of our research programs, and more generally for science in our respective countries.
