Regulatory sequences in sigma 54 localise near the start of DNA melting.

Transcription initiation by the enhancer-dependent sigma(54) RNA polymerase holoenzyme is positively regulated after promoter binding. The promoter DNA melting process is subject to activation by an enhancer-bound activator protein with nucleoside triphosphate hydrolysis activity. Tethered iron chelate probes attached to amino and carboxyl-terminal domains of sigma(54) were used to map sigma(54)-DNA interaction sites. The two domains localise to form a centre over the -12 promoter region. The use of deletion mutants of sigma(54) suggests that amino-terminal and carboxyl-terminal sequences are both needed for the centre to function. Upon activation, the relationship between the centre and promoter DNA changes. We suggest that the activator re-organises the centre to favour stable open complex formation through adjustments in sigma(54)-DNA contact and sigma(54) conformation. The centre is close to the active site of the RNA polymerase and includes sigma(54) regulatory sequences needed for DNA melting upon activation. This contrasts systems where activators recruit RNA polymerase to promoter DNA, and the protein and DNA determinants required for activation localise away from promoter sequences closely associated with the start of DNA melting.

The C-terminal 12 amino acids of sigma(N) are required for structure and function.

The sigma(N) protein is an alternative sigma subunit of bacterial RNA polymerase. We investigated the role of a 12-amino-acid "tail" at the C-terminus of Klebsiella pneumoniae sigma(N), which was predicted to be largely surface-exposed and to be mostly loop (that is not alpha-helical or beta-strand). Deletion of this tail from N-terminal hexahistidine-tagged sigma(N) led to loss of sigma(N)-dependent transcription activity in vivo. We overexpressed and purified this deletion-mutant protein for in vitro characterization. The purified deleted protein showed decreased RNA polymerase core- and DNA-binding activities compared to the full-length protein and transcription activity was greatly impaired. Furthermore, evidence from circular dichroism and protease digestion experiments together suggested that deletion of the C-terminus tail resulted in a loss of conformational constraint in the protein. We discuss a possible structural role for the 12 amino acids at the C-terminus of sigma(N).

Involvement of the sigmaN DNA-binding domain in open complex formation.

sigmaN (sigma54) RNA polymerase holoenzyme closed complexes isomerize to open complexes in a reaction requiring nucleoside triphosphate hydrolysis by enhancer binding activator proteins. Here, we characterize Klebsiella pneumoniae sigmaN mutants, altered in the carboxy DNA-binding domain (F354A/F355A, F402A, F403A and F402A/F403A), that fail in activator-dependent transcription. The mutant holoenzymes have altered activator-dependent interactions with promoter sequences that normally become melted. Activator-dependent stable complexes accumulated slowly in vitro (F402A) and to a reduced final level (F403A, F402A/F403A, F354A/F355A). Similar results were obtained in an assay of activator-independent stable complex formation. Premelted templates did not rescue the mutants for stable preinitiation complex formation but did for deleted region I sigmaN, suggesting different defects. The DNA-binding domain substitutions are within sigmaN sequences previously shown to be buried upon formation of the wild-type holoenzyme or closed complex, suggesting that, in the mutants, alteration of the sigmaN-core and sigmaN-DNA interfaces has occurred to change holoenzyme activity. Core-binding assays with the mutant sigmas support this view. Interestingly, an internal deletion form of sigmaN lacking the major core binding determinant was able to assemble into holoenzyme and, although unable to support activator-dependent transcription, formed a stable activator-independent holoenzyme promoter complex on premelted DNA templates.

Two domains within sigmaN (sigma54) cooperate for DNA binding.

The sigma-N (sigmaN) subunit of the bacterial RNA polymerase is a sequence specific DNA-binding protein. The RNA polymerase holoenzyme formed with sigmaN binds to promoters in an inactive form and only initiates transcription when activated by enhancer-binding positive control proteins. We now provide evidence to show that the DNA-binding activity of sigmaN involves two distinct domains: a C-terminal DNA-binding domain that directly contacts DNA and an adjacent domain that enhances DNA-binding activity. The sequences required for the enhancement of DNA binding can be separated from the sequences required for core RNA polymerase binding. These results provide strong evidence for communication between domains within a transcription factor, likely to be important for the function of sigmaN in enhancer-dependent transcription.