The cya, crp and rho gene products modulate the expression of a wide variety of bacterial and bacteriophage genes or operons. In order to understand the regulatory processes, we are studying the structure, expression and activity of these genes and gene products. We have previously shown that the protein products of the cya, crp, and rho genes are autogenously regulated. In CRP, cyclic AMP binding to the amino-terminal domain induces an allosteric transition which changes the DNA binding property of the carboxy domains. We have isolated mutants in the crp gene called crpu, which make CRP functional in the absence of cyclic AMP. The mutations responsible for the CRPu phenotype cause substitutions by amino acids with bulkier side chains in the D Alpha-helix of the protein's carboxy domain, near the hinge which connects the carboxy to the amino domain. Apparently, the mutant CRP's have assumed a conformation that is normally evoked by cAMP binding. We have isolated intragenic suppressors of crpu that restore the need for cAMP. These mutants define precisely the amino acids and thus the parts of CRP which interact specifically to cause the allosteric shift. We have shown previously by pulse-labeling of RNA and by DNA-RNA hybridization, as well as by operon fusion analysis, that the rho gene appears to be regulated by cAMP and CRP at the levels of transcription and translation. By studying the system in vitro, using coupled and non-coupled transcription/translation systems, we have been unable to show a direct CRP-cAMP effect on rho expression. Since the effect appears indirect, we are currently using a genetic approach to identify the factors involved.
