Title: Modulation of promoter escape by sigma-core interactions of the bacterial RNA polymerase holoenzyme
Description: The bacterial RNA polymerase (RNAP) core enzyme contains all of the catalytic machinery required for RNA chain elongation during transcription. However, it must associate with a σ factor to form an RNAP holoenzyme species that is competent to recognize promoter sequences and initiate transcription. The primary a factor of Escherichia coli, σ 70 , directs transcription from promoters typically defined by two conserved sequence elements, the -10 and -35 elements, that are separated by ∼17 base pairs (bp). All primary σ factors share four regions of conserved sequence (regions 1-4; σ 1 -σ 4 ) that are connected by intervening sequences of variable size, and regions 2 and 4 contain DNA-binding domains that recognize the -10 and -35 elements, respectively. σ 70 forms extensive contacts with the core enzyme in the context of the RNAP holoenzyme. The interaction between σ 70 region 2(σ 2 ) and the β' coiled-coil, in particular, is required for σ 2 to make sequence-specific contacts with bases of the non-template strand of the promoter -10 element in the open promoter complex. σ 2 can also contact the non-template strand of -10 element-like sequences during early elongation, mediating promoter-proximal pauses. As during open complex formation, establishment of sequence-specific contacts between σ 2 and the -10-like pause-inducing sequence depends on the σ 2 /β' coiled-coil interaction. Thus, the σ 2 /β' coiled-coil interaction hinders escape from promoter (and promoter-like pause-inducing sequences).

This dissertation describes a previously uncharacterized interaction between the σ 70 non-conserved region (NCR), which is located between σ 1 , and σ 2 , and a domain of the β' subunit called the "sigma NCR interaction domain" (S NCR ID). In contrast to the σ 2 /β' coiled-coil interaction, the σ 70 NCR/β' S NCR ID interaction facilitates escape from promoters (and promoter-like pause-inducing sequences). Expression microarray analysis of a strain in which the σ 70 NCR/β' S NCR ID interaction is disrupted (via mutation) was used to search for promoters regulated by σ-dependent pausing. Disruption of this interaction was found to reduce in vivo transcript levels of nanC, which encodes a sialic acid-specific transporter. σ 70 mediates a pause at the nanC promoter, and this pause may be important for regulation of nanC expression by the transcription activator Crp.