the e. coli anti-sigma factor rsd studies on the specificity and regulation of its expression大肠杆菌anti-sigma因素为研究特异性和监管的表达式.pdf

The E. coli Anti-Sigma Factor Rsd: Studies on the Specificity and Regulation of Its Expression Nina Hofmann, Reinhild Wurm, Rolf Wagner* ¨ ¨ ¨ ¨ Institut fur Physikalische Biologie, Heinrich-Heine-Universitat Dusseldorf, Dusseldorf, Germany Abstract Background: Among the seven different sigma factors in E. coli s70 has the highest concentration and affinity for the core RNA polymerase. The E. coli protein Rsd is regarded as an anti-sigma factor, inhibiting s70-dependent transcription at the onset of stationary growth. Although binding of Rsd to s70 has been shown and numerous structural studies on Rsd have been performed the detailed mechanism of action is still unknown. Methodology/Principal Findings: We have performed studies to unravel the function and regulation of Rsd expression in vitro and in vivo. Cross-linking and affinity binding revealed that Rsd is able to interact with s70, with the core enzyme of RNA polymerase and is able to form dimers in solution. Unexpectedly, we find that Rsd does also interact with s38, the stationary phase-specific sigma factor. This interaction was further corroborated by gel retardation and footprinting studies with different promoter fragments and s38- or s70-containing RNA polymerase in presence of Rsd. Under competitive in vitro transcription conditions, in presence of both sigma factors, a selective inhibition of s70-dependent transcription was prevailing, however. Analysis of rsd expression revealed that the nucleoid-associated proteins H-NS and FIS, StpA and LRP bind to the regulatory region of the rsd promoters. Furthermore, the major promoter P2 was shown to be down-regulated in vivo by RpoS, the stationary phase-specific sigma factor and the transcription factor DksA, while induction of the stringent control enhanced rsd promoter activity. Most no