Molecular mechanisms of transcription and transcriptional regulation, including elongation control of virulence genes in proteobacteria.
The focal point of the research in our lab is RNA polymerase (RNAP), the enzyme that is responsible for the first step in gene expression, mRNA synthesis. RNAP accomplishes this task during the transcription cycle that is composed of three major steps: initiation, elongation, and termination. All these steps are subject to elaborate control by numerous regulatory proteins and small effectors. RNAP is also an attractive target for antibacterial drugs. Using a combination of biochemical, genetic, and structural (in collaboration with Dr. Vassylyev’s Lab at UAB) approaches, we are currently working on several projects:
Substrate selection by RNAP
To transmit genetic information from genome to proteome in undistorted form, RNAP must synthesize the nascent RNA with high fidelity. Fidelity mechanisms are well-studied in DNA polymerases, and to a lesser extent in single-subunit (phage T7) RNAPs . However, the mechanism of substrate selection by multi-subunit enzymes has not been studied. We are utilizing structure-based mutagenesis to elucidate this mechanism of correct nucleotide selection, and have already obtained a set of RNAP variants with altered substrate selection properties.
Mechanism and regulation of RNA chain elongation and termination
Tth RNAP elongation complex
The rate of transcription is determined by the nucleic acid signals that slow transcription. These signals serve as regulatory checkpoints at which RNAP could be modified by action of auxiliary factors, and therefore determine the gene expression patterns in all organisms. We want to determine how certain DNA and RNA sequences trigger RNAP isomerization into an un-reactive, slow state, which is characterized by a dramatic decrease in the rate of nucleotide addition, and is a likely target for elongation factors (such as NusA, NusG and RfaH). We study how RNAP itself recognizes transcription roadblocks and how auxiliary factors affect its behavior.
RfaH, an elongation enhancer protein
Efficient synthesis of long messages relies on transcription factors that allow RNAP to overcome transcription roadblocks. RfaH is a bacterial antitermination factor that enables RNAP to transcribe through long polycistronic operons encoding toxins, antibiotics, capsules, lipopolysaccharide core, and F-pili, all of which are molecules that contribute to bacterial pathogenesis. We are studying the molecular mechanism by which RfaH “switches” RNAP into a highly processive state, pursuing determination of the X-ray structure of RfaH, characterizing the RfaH regulon in E. coli , and conducting the comparative analysis of RfaH orthologs from different bacteria (Y. enterocolitica, V. cholerae, K. pneumoniae, etc.).
Molecular mechanisms of transcriptional inhibitors action on RNAP
Rifabutin and rifapentin bound to Tth RNAP holoenzyme, solution.
Inhibitors of bacterial RNAP are used as antibiotics to treat bacterial infections and in research to gain insights into molecular mechanisms that regulate transcription. We are working on the mechanism of RNAP inhibition by rifamycins, tagetitoxin, and CBRs. We perform detailed analysis of the mechanism of action of these inhibitors by a combination of genetic and biochemical techniques and collaborate with Dr. Vassylyev to obtain high-resolution X-ray structures of the Thermus thermophilus RNAP in complex with these drugs. We plan to use the collected data for design ofnovel antibiotics.
Regulation of transcription through RNAP secondary channel
RNAP secondary channel postulated to facilitate delivery of substrate NTPs to the active site appears to facilitate access of other small molecules and auxiliary factors to the catalytic center of the enzyme. Alarmone ppGpp, inhibitor of chloroplast development tagetitoxin, cleavage factors GreA and GreB, and their structural analog DksA all have been recently added to the growing list of transcriptional regulators utilizing secondary channel as the only accessible venue connecting RNAP active site to the surface of the enzyme. Presently we collaborate with Dr. Vassylyev’s Laboratory at UAB in elucidating the mechanisms by which these and other factors regulate activity of the RNA polymerase.