Optimization of recombinant Mycobacterium tuberculosis RNA polymerase expression and purification.
Mycobacterium tuberculosis, the human pathogen that causes tuberculosis, warrants enormous attention due to the emergence of multi drug resistant and extremely drug resistant strains. RNA polymerase (RNAP), the key enzyme in gene regulation, is an attractive target for anti-TB drugs. Understanding the structure-function relationship of M. tuberculosis RNAP and the mechanism of gene regulation by RNAP in conjunction with different σ factors and transcriptional regulators would provide significant information for anti-tuberculosis drug development targeting RNAP. Studies with M. tuberculosis RNAP remain tedious because of the extremely slow-growing nature of the bacteria and requirement of special laboratory facility. Here, we have developed and optimized recombinant methods to prepare M. tuberculosis RNAP core and RNAP holo enzymes assembled in vivo in Escherichia coli. These methods yield high amounts of transcriptionally active enzymes, free of E. coli RNAP contamination. The recombinant M. tuberculosis RNAP is used to develop a highly sensitive fluorescence based in vitro transcription assay that could be easily adopted in a high-throughput format to screen RNAP inhibitors. These recombinant methods would be useful to set a platform for M. tuberculosis RNAP targeted anti TB drug development, to analyse the structure/function of M. tuberculosis RNAP and to analyse the interactions among promoter DNA, RNAP, σ factors, and transcription regulators of M. tuberculosis in vitro, avoiding the hazard of handling of pathogenic bacteria.
Department of Chemistry, Bose Institute, 93/1 APC Road, Kolkata 700009, India. Electronic address: rajdeepbenerjy@gmail.com.
Rajdeep
R
Banerjee
Department of Chemistry, Bose Institute, 93/1 APC Road, Kolkata 700009, India. Electronic address: paula_rud@rediffmail.com.
Paulami
P
Rudra
Department of Chemistry, Bose Institute, 93/1 APC Road, Kolkata 700009, India. Electronic address: prajapatichem2009@gmail.com.
Ranjit Kumar
RK
Prajapati
Department of Chemistry, Bose Institute, 93/1 APC Road, Kolkata 700009, India. Electronic address: shreya.sengupta0@gmail.com.
Shreya
S
Sengupta
Department of Chemistry, Bose Institute, 93/1 APC Road, Kolkata 700009, India. Electronic address: jayanta@jcbose.ac.in.
Jayanta
J
Mukhopadhyay
D000995:Antitubercular Agents / Q000494:pharmacology; D003001:Cloning, Molecular / Q000379:methods; D012321:DNA-Directed RNA Polymerases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D015195:Drug Design; D004926:Escherichia coli / Q000201:enzymology; D006801:Humans; D008826:Microbial Sensitivity Tests / Q000379:methods; D058990:Molecular Targeted Therapy / Q000379:methods; D009169:Mycobacterium tuberculosis / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012808:Sigma Factor / Q000378:metabolism; D014158:Transcription, Genetic
D000995:Antitubercular Agents; D019384:Nucleic Acid Synthesis Inhibitors; D011994:Recombinant Proteins; D012808:Sigma Factor; D012321:DNA-Directed RNA Polymerases
Mycobacterium tuberculosis; Omega; RNA polymerase; Sigma factor; Transcription