The sigma subunit of eubacterial RNA polymerase is essential for initiation of transcription at promoter sites. Sigma factor directs the RNA polymerase core subunits (alpha2betabeta’) to the promoter consensus elements and thereby confers selectivity for transcription initiation. The N-terminal domain (region 1.1) of Escherichia coli sigma70 has been shown to inhibit DNA binding by the C-terminal DNA recognition domains when sigma is separated from the core subunits. Since DNA recognition by RNA polymerase is the first step in transcription, it seemed plausible that region 1 might also influence initiation processes subsequent to DNA binding. This study explores the functional roles of regions 1.1 and 1.2 of sigma70 in transcription initiation. Analysis in vitro of the transcriptional properties of aseries of N-terminally truncated sigma70 derivates revealed a critical role for region 1.1 at several key stages of initiation. Deletion of the first 75 to 100 amino acids of sigma70 (region 1.1) resulted in both a slow rate of transition from a closed promoter complex to a DNA-strand-separated open complex, as well as a reduced efficiency of transition from the open complex to a transcriptionally active open complex. These effects were partially reversed by addition of a polypeptide containing region 1.1 in trans. Therefore, region 1.1 not only modulates DNA binding but is important for efficient transcription initiation, once a closed complex has formed. A deletion of the first133 amino acids which removes both regions 1.1 and 1.2 resulted in arrest of initiation at the earliest closed complex, suggesting that region 1.2 is required for open complex formation. Mutagenesis of region 1.1 uncovered a mechanistically important role for isoleucine at position 53 (I53). Substitution of I53 with alanine created a sigma factor that associated with the core subunits to form holoenzyme, but the holoenzyme was severely deficient for promoter binding. The I53A phenotype was suppressed in vivo by truncation of five amino acids from the C-terminus of sigma70. These observations are consistent with a model in which sigma70 I53A fails to undergo a critical conformational change upon association with the core subunits, which is needed to expose the DNA binding domains and confer promoter recognition capability upon holoenzyme. To understand the basis of the autoinhibitory properties of the sigma70 N-terminal domain, in the absence of core RNA polymerase, a preliminary physical assessment of the interdomain interactions within the sigma70 subunit was launched. Results support a model in which N-terminal amino acids are in close proximity to residues in the C-terminus of the sigma70 polypeptide.
The Role of Conserved Region 1 of Escherichia coli Sigma-70 in Transcription Initiation