RNA BIOSYNTHESIS IN ESCHERICHIA COLI

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A study of structure-function relationships in DNA-dependent RNA polymerase (RNAP) of Escherichia coli is proposed which will use a combination of protein-chemical and enzymological approaches. The ultimate goal of this study is the understanding of RNAP basic function and regulation in molecular detail, and the uncovering of the underlying structural determinants. The emphasis of this project is on topology of the ternary transcription complex as it propagates along DNA. We will test the model that the movement of the complex occurs in the "inchworm" fashion whereby the enzyme compresses and extends with the amplitude of several nucleotides. Understanding of the mechanism of RNA polymerase propagation will provide clues to the design of this enzyme as a multifunctional molecular machine which serves as the target to factors that regulate gene expression. The proposed study will be focused on the RNA component of the ternary complex. Three principal approaches will be used. First we intend to use crosslinkable affinity probes incorporated into defined positions in the transcript to fix RNA in the complex, and then to study such complex enzymatically. The impediment to the normal RNA chain extension resulting from the crosslink will help understand the mechanism of RNAP propagation. The second approach employs the GreA protein, a novel transcript cleavage factor that we discovered, as a probe of RNA topology in the ternary complex. GreA cleaves the nascent transcript exposed in the vicinity of the enzyme's catalytic center. From the changes of the cleavage pattern that take place during complex propagation we expect to learn about the mechanism of the enzyme movement. Finally, we propose to identify amino acids involved in contacts with RNA in the ternary complex, and the length of DNA-RNA hybrid region by means of affinity labeling with probes incorporated into the transcript followed by mapping the adducts using methods of protein and nucleic acid chemistry.