| In bacteria, all genes are transcribed by a single multisubunit entity called RNA polymerase (RNAP). The enzymatic core of RNAP contains all of the requisite machinery for RNA synthesis. Nevertheless, in order to recognize specific promoters and initiate transcription, the core enzyme must associate with a σ factor, which makes direct contact with conserved promoter elements. The primary σ factor in E. coli, σ 70, is responsible for the expression of the majority of genes in exponentially growing cells. As well as playing a critical role; in promoter recognition, σ70 is well-characterized as a target for proteins that regulate transcription initiation.; Until recently, it has been thought that σ70 is released from the core enzyme during the transition from the initiation to the elongation phase of transcription. However, emerging evidence suggests that promoter escape may not necessarily require the complete release of σ 70 from core. In fact, models based on recently obtained structural data posit a staged (rather than complete) displacement of σ 70 from the core enzyme during the transition from initiation to elongation.; This thesis illustrates how interactions between regulators of transcription and the σ70 subunit of RNAP can be exploited during different stages of the transcription process. In particular, I have characterized the interactions of two transcription regulators with the C-terminal domain (conserved region 4) of σ70. One of these regulators, the bacteriophage λ cI protein, is an activator of transcription initiation. In Chapter 21 demonstrate the use of a genetic assay and structural modeling to implicate two amino acid residues within region 4 of σ70 in the process of λcI-mediated activation. The other regulator that I have studied, the bacteriophage λ Q antiterminator protein, acts during elongation. In Chapter 3 I demonstrate that λQ contacts region 4 of σ70 when λQ engages a paused elongation complex. I show further that this engagement results in a conformation of the RNAP holoenzyme that requires the partial release of σ70 from core. These results demonstrate that σ 70 can be a target for a regulator that exerts its effect on transcription at a stage after the initiation process and provide evidence for the staged displacement of σ70 during the transition from transcription initiation to transcription elongation. |
