Dissecting the interaction between Escherichia coli 6S RNA and the RNA polymerase sigma70 subunit

The Escherichia coli 6S RNA is a small RNA which forms a specific, stable complex with RNA Polymerase (RNAP) containing sigma 70 (Esigma70). sigma70 is the RNAP subunit that conveys specificity to the enzyme by making direct contact with the -35 and -10 hexamers that define sigma70-dependent promoters. sigma 70 contains four conserved regions that each play a role in contacting promoter elements, including region 2 binding to the -10 hexamer and region 4 binding to the -35 hexamer.;6S RNA forms a phylogenetically conserved secondary structure that resembles promoter DNA in the open complex conformation, and this secondary structure is essential for the interaction with Esigma70. By binding Esigma70, 6S RNA inhibits transcription from a subset of sigma 70-dependent promoters by directly competing with promoter DNA for binding to Esigma70. 6S RNA is found within the Esigma 70 active site, suggesting that the 6S RNA-Esigma70 contacts overlap the location on Esigma70 that binds promoter DNA.;My thesis research was to further understand the 6S RNA-Esigma 70 interaction by analyzing sigma70 mutants to elucidate a 6S RNA binding surface onsigma70 (within Esigma 70). I have shown that sigma70 region 4.2, which contacts the -35 hexamer through a helix-turn-helix structural motif, is critical for binding 6S RNA as well. sigma70 subunits with single alanine substitutions identified residues within sigma70 region 4.2 that are important for binding 6S RNA. These studies showed that the 6S RNA binding surface on sigma70 region 4.2 is distinct but overlapping with the DNA binding site. Furthermore, the 6S RNA binding surface is defined by multiple positively-charged residues, all of which contribute significantly to binding. My research has defined an RNA binding interaction with sigma70 region 4.2 that is divergent from DNA binding, which is likely to be one of many contacts between 6S RNA and Esigma70.