coli and E. chaffeensis σ70 subunits of RNAP share high degree of homology. Transcriptional inhibition of the enzyme by the anti- σ70monoclonal antibody and rifampin, a potent inhibitor of prokaryotic RNAP [27, 38], demonstrates that the in vitro transcriptional activity in our study was due to the isolated E. chaffeensis RNAP. Transcriptional profiles depicting salt tolerance of purified

enzymes have been described for prokaryotes, such as, C. trachomatis and www.selleckchem.com/products/Adrucil(Fluorouracil).html E. coli [20, 39]. In E. coli, transcription of a σ70-regulated promoter decreases dramatically between 100 mM and 150 mM potassium acetate [39], whereas σ66-dependent promoter activity of Chlamydia is completely inhibited at 400 mM concentration [20]. The purified E. chaffeensis RNAP, reported in this study, also showed a similar range of salt tolerance as observed for other bacterial σ70 dependent RNAPs.

For example, the enzyme showed optimum transcriptional activity at 80 mM sodium chloride, a slight difference from the optimal 50 mM concentration reported for the R. prowazekii RNAP [27]. The minor differences in the salt tolerance properties may be unique to E. chaffeensis RNAP. Previous studies suggest that RNAP fractions purified by heparin-agarose chromatography methods are typically about 30% saturated with the major sigma subunit [20]. Thus the C59 wnt manufacturer presence of free core enzymes in the preparation allows reconstitution studies or saturation with recombinant sigma factors to enhance transcription in vitro. Thus we prepared a purified recombinant E. chaffeensis σ70 subunit and used for assessing transcriptional activity by Non-specific serine/threonine protein kinase saturation of the native enzyme or by reconstitution with E. coli core enzyme. Saturation of the purified RNAP with the recombinant subunit resulted

in enhanced transcriptional signals. Reconstitution of E. coli core enzyme with E. chaffeensis recombinant σ70 subunit had similar salt sensitivities to that of purified E. chaffeensis RNAP before and after saturating with the recombinant subunit. These data are consistent with earlier reports indicating that purified C. psittacci σ66 was effective in stimulating transcription by C. trachomatis and C. psittaci RNAP preparations [32] and highlights that E. coli core enzyme reconstituted with E. chaffeensis sigma factor offers an alternative approach to in vitro characterization of E. chaffeensis promoters as described for C. trachomatis [20, 33]. Previously, we and others reported the use of E. coli system in characterizing the promoters of E. chaffeensis [25, 40]. The current study offers an additional advantage over the E. coli system in that it uses E. chaffeensis RNAP or E. coli core enzyme with E. chaffeensis recombinant σ70. Regulation of gene transcription in prokaryotes involves a complex network and is controlled at the stage of RNA synthesis in which transcription factors (TFs) are key components [41, 42].