Modulation of promoter escape by sigma-core interactions of the bacterial RNA polymerase holoenzyme

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.