Mechanism of stimulation of sigma 54 and sigma 70 RNA polymerase holoenzymes by nucleotides

Regulation of transcription initiation is needed for proper gene expression in the cell. Two families of sigma factors are involved in bacterial gene expression, sigma 54 and sigma 70. Sigma 54 RNA polymerase (RNAP) needs both an activator and ATP for transcription. Sigma 70 RNA polymerase can transcribe without activator, but can still be regulated. An example of a highly regulated sigma 70 promoter is the ribosomal promoter, which surprisingly, appears to be regulated by nucleotide rather than macromolecule effectors.; The focus of this dissertation is to better understand the roles of nucleotides in regulation of these sigma RNA polymerases. For the sigma 54 system, it is shown that ATP and activator are needed to change the conformation of sigma 54 to expose the single strand DNA binding determinants needed to open promoter DNA. The C3 and C7 regions of the central domain of these activators assure proper regulation by inhibiting premature oligomerization of dimers.; In the sigma 70 system, the mechanism of regulation of ribosomal RNA promoters by nucleotides was investigated. It was shown that lack of initiating nucleotides inhibits transcription because polymerase fails to fully isomerize when the concentration of initiating nucleotides is low. Initiating nucleotides stimulate rRNA transcription by increasing the population of complexes that isomerize and not by stabilizing the half-life of transcription complexes. In addition to a stimulatory role of the +1 nucleotide-binding site, the +2 site also plays a stimulatory role in transcription at these promoters. Using cross-linking, we see that in the presence of nucleotides, RNA polymerase engages the template strand better making RNAP more prepared for transcription initiation. A structure-based model for this regulation is proposed. These findings help elucidate the regulation of transcription initiation from these two families of RNA polymerases.