B. pertussis infects the upper respiratory tract by adhering to ciliated epithelial cells and releasing toxins. The virulence genes that encode these proteins are regulated by the BvgS-BvgA two-component system. The response regulator BvgA binds to the promoter region of all known B. pertussis virulence genes to activate their transcription during infection. These genes include bipA, fha, ptx, prn, cya, bvgR, bvgA, and the fim genes. The fim2 and fim3 genes encode different surface proteins that facilitate adhesion of the bacterium to epithelial cells in the human respiratory tract. The promoter regions of fim3, fim2, and a paralogous gene fimX, share high sequence homology, suggesting a similar mechanism of regulation. ? ? The sigma70 subunit of RNA polymerase (RNAP) is the specificity factor that recognizes promoter elements. Residues within sigma70 regions 2, 3, and 4 interact with sequences in the -35 element, the extended -10 sequence (positions -15, -14), and sequence in the -10 element, respectively. Promoters which have poor sigma70-dependent elements typically require other factors to promote transcription initiation. There are three well-established mechanisms of bacterial activation. In Class I and Class II activation, the activators improve the interaction of RNAP sigma70 with the -35 element. In a third class of activation, termed sigma appropriation, the -35 region is occupied by a transcriptional activator and RNAP is partially displaced from thi region of the DNA. To date, sigma appropriation has only been characterized in bacteriophage T4. ? ? Experiments in the laboratory of Dr. Scott Stibitz (FDA) using a site-specific cleavage reagent have mapped the activator BvgA to the -35 region of the fim3 promoter. This binding pattern is incompatible with the position of RNAP as described in Class I and Class II activation, suggesting that the activation of fim3 may involve sigma appropriation, or it may involve a novel mechanism of prokaryotic gene activation. ? ? To investigate how the fim genes are regulated by BvgA, we are collaborating with the laboratory of Dr. Stibitz. We have identified the in vivo transcription start sites of fim3, fim2, and fimX by primer extension. The fim3 gene contains an extended -10 promoter element whereas fim2 and fimX lack this element. All three genes have the -10 element but, lack a recognizable -35 element, suggesting that this region may be unimportant for fim activity. However, studies in other labs with different extended -10 promoters have shown that sigma70 region 4 can still occupy the DNA near -35 in the absence of a canonical -35 element.? ? Using in vitro transcription experiments, we have found that that the fim3 promoter does not use the typical interactions with sigma70 region 4. We have shown this with three independent methods. First, fim3 does not need the sigma70 resides normally required for interaction with the -35 promoter DNA. Second, a protein that disrupts sigma70 -35 DNA interaction (bacteriophage T4 AsiA) does not inhibit BvgA activation of fim3. Third, the far C-temrinal region of sigma70, which is normally required for contact with the beta-flap portion of RNAP, is not required for BvgA activation of fim3. Our results suggest that sigma70 region 4 is less important for fim3 activation than at a typical gene. Taken together, these findings are consistent with a model in which BvgA and sigma70 cooperate or compete to bind the DNA at the -35 element in order to activate the fim genes.
| Decker, Kimberly B; Hinton, Deborah M (2009) The secret to 6S: regulating RNA polymerase by ribo-sequestration. Mol Microbiol 73:137-40 |
