AUTOREGULATION AND GLOBAL CONTROL OF THE CRP OPERON

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Cyclic AMP is a key compound in many organisms involved in regulating numerous aspects of normal and abnormal cellular growth and differentiation. In most systems this control is mediated by a cyclic AMP binding protein. This protein is necessary for the biological function of cyclic AMP and, in some cases, the protein is also involved in regulating cyclic AMP synthesis. A knowledge of how the levels of the cyclic AMP binding protein is controlled is an important aspect of understanding how this key control element functions in the cell. We have begun a study of the regulation of the cyclic AMP binding protein (CRP) in E. coli. Our initial investigations suggest a unique mechanism for control of expression of the crp operon. This mechanism involves the activation by cyclic AMP and CRP of a divergently transcribed RNA that initiates close to the start of crp transcription but on the opposite strand. This RNA blocks the synthesis of crp mRNA. We wish to continue these studies using in vitro and in vivo techniques to prove that this novel mechanism regulates crp expression and to understand the molecular basis for its action. In vitro work will include site directed mutagenesis, the effect of the divergent RNA on transcription and experiments to test if the divergent RNA forms a duplex with the 5' end of crp mRNA. In vivo experiments will focus on the use of cloned fragments of the crp and divergent promoter to assess the role of the divergent transcription in crp regulation in vivo.