E. coli RNA polymerase binds promoter DNA to a channel on its surface and is able to melt about 12 base pairs of the promoter DNA to form a strand separated or """"""""open"""""""" complex. On the pathway to formation of this complex, several distinct intermediate complexes have been recognized. The transition between two of them likely involves a major conformational change in the RNA polymerase, triggering the nucleation of strand separation in the - 10 region, from where basepair disruption propagates in downstream direction. Three additional regions of sequence-specific interaction between E. coli RNA polymerase and promoters have been recognized: a TG dinucleotide just upstream of the -10 element, the -35 region and the UP element at -40 through -60. We propose to investigate several aspects of the interaction of RNA polymerase with nucleic acids, and the role of sigma factor in the process. Using several different approaches we will better define the role of the TG, -35 and UP sequences in facilitating open complex formation. We will continue our studies on the interaction of RNA polymerase with single stranded (ss) DNA, and characterize the conformational change in RNA polymerase that accompanies ss DNA binding. We will also initiate the characterization of the thermodynamics of the interaction between ss DNA and the RNA polymerase of a thermophile (optimally active at 55xC). The group of transcription initiation factors known as sigma factors is responsible for the interaction with promoter DNA at the -10 and -35 regions, the TG sequence and ss DNA in the open complex. We will determine he role of highly conserved residues of sigma factor in open complex formation by introduction of alanine substitutions in a region spanning conserved regions 2.2, 2.3 and 2.4. Selected substitutions will be combined to generate sigma factors severely defective in the DNA melting step. These will be useful in probing the process of nucleation of DNA strand separation. In addition we will study RNA polymerase containing sigma32, which differs from sigma70 in conserved region 2.3, and also in the promoter sequence it recognizes.
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