The RpoN‐box motif of the RNA polymerase sigma factor σ^N plays a role in promoter recognition

Abstract

The RNA polymerase sigma factor sigma N (sigma 54) is characterized by the presence, near the C-terminal end of the protein, of a highly conserved sequence of 10 amino acids (ARRTVAKYRE) that has been termed the RpoN box. In order to examine the function of this motif, which is predicted to adopt an alpha-helical structure, we have isolated a number of mutations that alter residues within the box and examined the properties of the sigma N derivatives encoded by them. Certain mutations that alter charged and potentially exposed residues within the motif result in transcriptionally inactive proteins with impaired promoter recognition but no impairment in core RNA polymerase binding. We therefore suggest that the RpoN box could play a direct or indirect role in recognition of the -24, -12 promoter consensus that is characteristic of sigma N-dependent genes.

Probing the assembly of transcription initiation complexes through changes in σ N protease sensitivity

“…Because holoenzyme binds promoters differently both quantitatively and qualitatively from free N (46,47) it is plausible that the predicted conformational changes result in altered DNA binding activity. In this regard the protection of E378 from V8 cleavage in the holoenzyme is particularly interesting, as it lies in a region involved specifically in DNA binding and intolerant to mutagenesis (15,24,26,27). In addition, region I influences DNA binding around the Ϫ12 element (15)(16)(17)47), suggesting that region I conformational changes could modify N -DNA interactions.…”

“…USA 94 (1997) 12147 turn-helix motif and the rpoN box. Both of these elements have been implicated in promoter recognition (26,27,37,41,42), so it is plausible that protection of E410͞414 and E431 results from their proximity to DNA. No reproducible differences were seen between holoenzyme and closed complexes with chymotrypsin or subtilisin digestion.…”

“…Next, residues 329-477 comprise the primary DNA binding determinant (15,23,24) and include a predicted helix-turn-helix motif (25) and the rpoN box, a characteristic element found in all N proteins. Mutations in either of these sequence elements can alter DNA binding (26,27). Finally, residues 180-306 contribute to DNA binding activity by enhancing binding of the minimal DNA binding domain (28).…”

Analysis of the architecture of the transcription factor σN (σ54) and its domains by circular dichroism

“…Our experiments do not address whether the 36ЊC transition in the structure of the full-length protein is simply fortuitously close to the physiological or whether it indicates a potential for readily accomplishing a conformational change relevant to function. Although the critical residues within the 425-477 deletion interval responsible for the markedly altered melting behaviour of the C-terminally truncated protein are not defined, the so-called RPON box, a conserved sequence of eight amino acids in the C-terminus of N proteins, is a candidate structural element in N (Cannon et al, 1995a;Merrick, 1993;Taylor et al, 1996). Since interactions between residues 329-477 and 180-306 are important for DNA-binding (Cannon et al, 1997) these interactions may be reflected in the altered melting transitions discussed above.…”

“…2A), a lack of tyrosine 461 may cause a reduction in the CD signal in the near-UV. Notably, tyrosine 461 is within the so-called 'RPON box', a conserved motif diagnostic of N (Merrick, 1993;Taylor et al, 1996). The 70-324 core-binding proteins exhibited a stronger aromatic spectrum than did N (Table 1 and Fig.…”