Mode of selectivity in cyclic AMP receptor protein-dependent promoters in Escherichia coli.

Escherichia coli cAMP receptor protein (CRP) controls more than 20 genes. There are significant differences in the promoter regions in these genes. Thus, an elucidation of the mechanism of CRP action requires knowledge about the mode of selectivity in these promoters. An earlier study [Heyduk, T., & Lee, J. C. (1990) Proc. Natl. Acad. Sci. U.S.A. 81, 1744-8] indicates that the CRP(cAMP)1 conformer exhibits the highest affinity for the lac PI site in the lac operon. It is conceivable that the CRP conformer that binds with the highest affinity to these other sites may not be CRP(cAMP)1. To investigate this possibility, the binding of CRP to nine CRP binding sites was studied as a function of cAMP concentration. The CRP binding sites employed in this investigation were chosen to represent the primary promoter sites from class I (lac site PI) and class II (sites PI of gal and crp) as well as secondary CRP binding sites (crp site PII and cat PII) to further understand the molecular mechanism of CRP in controlling the transcription of these bacterial genes. The affinity of CRP for three synthetic CRP binding sites was also examined to explore the contribution of the inverted repeat region and sequences surrounding the recognition motifs. The synthetic sequences are gallac which contains the lac recognition motifs in the background of gal, modified cat PII which contains an 8-base pair spacer between the recognition motifs rather than the 7-base pair sequence naturally found in cat PII, and a random sequence that has no known similarity to any CRP binding site found in nature. The apparent affinities of these sequences for CRP were quantitatively determined to be biphasic in their cAMP dependence. The CRP(cAMP)1 conformer was found to have the highest affinity for all of the DNA sequences examined. No specific affinity was observed for these sequences with free CRP and CRP(cAMP)2. The affinity of CRP for DNA was sequence-dependent and increased in the following order: random < cat site PII, modified cat site PII, crp sites PI and PII < gal site PI < lac site PI < gallac. These results indicate that the entire CRP binding site sequence and its natural variability provide information to CRP. These promoter sites which appear to have different mechanisms at the molecular level are transcriptionally controlled by the same CRP conformer, CRP(cAMP)1. Thus, the regulation of transcription by CRP is more subtle than choosing different conformational forms of CRP. Using "physiological" concentrations of various components, a computer simulation study was conducted to illustrate the possible consequences of the thermodynamic parameters determined in this study. It is evident that the promoters of protein systems regulating the transport and metabolism of carbohydrates are responsive to low cAMP concentrations. However, the promoter for controlling the expression of CRP is highly regulated by the fluctuation of cAMP concentration.