A Transcript Cleavage Factor of Mycobacterium tuberculosis Important for Its Survival

Abstract

After initiation of transcription, a number of proteins participate during elongation and termination modifying the properties of the RNA polymerase (RNAP). Gre factors are one such group conserved across bacteria. They regulate transcription by projecting their N-terminal coiled-coil domain into the active center of RNAP through the secondary channel and stimulating hydrolysis of the newly synthesized RNA in backtracked elongation complexes. Rv1080c is a putative gre factor (MtbGre) in the genome of Mycobacterium tuberculosis. The protein enhanced the efficiency of promoter clearance by lowering abortive transcription and also rescued arrested and paused elongation complexes on the GC rich mycobacterial template. Although MtbGre is similar in domain organization and shares key residues for catalysis and RNAP interaction with the Gre factors of Escherichia coli, it could not complement an E. coli gre deficient strain. Moreover, MtbGre failed to rescue E. coli RNAP stalled elongation complexes, indicating the importance of specific protein-protein interactions for transcript cleavage. Decrease in the level of MtbGre reduced the bacterial survival by several fold indicating its essential role in mycobacteria. Another Gre homolog, Rv3788 was not functional in transcript cleavage activity indicating that a single Gre is sufficient for efficient transcription of the M. tuberculosis genome.

Inhibition of Mycobacterium tuberculosis RNA Polymerase by Binding of a Gre Factor Homolog to the Secondary Channel

“…The Rv3788 gene from M. tuberculosis was cloned in pET20b vector with a C-terminal His tag between the NdeI and HindIII sites (9). E. coli BL21 cells with pET20b-Rv3788 were grown to an optical density at 600 nm (OD 600 ) of 0.6 and induced with 0.3 mM isopropyl-␤-D-thiogalactopyranoside (IPTG).…”

“…MSMEG_6292 was PCR amplified from M. smegmatis mc 2 155 genomic DNA using the primers listed in Table S1 in the supplemental material and cloned into pET20b (at NdeI and HindIII sites) with a C-terminal His tag and purified by following the same method as that for His-Rv3788. M. tuberculosis and M. smegmatis Gre (MtbGre and MsGre, respectively) proteins were purified by following the methods described earlier (9). M. tuberculosis and M. smegmatis RNAP (MtbRNAP and MsRNAP, respectively), which were used for in vitro transcription assays and protein-protein interaction studies, were purified by following the method described earlier (9,10).…”

“…M. tuberculosis and M. smegmatis Gre (MtbGre and MsGre, respectively) proteins were purified by following the methods described earlier (9). M. tuberculosis and M. smegmatis RNAP (MtbRNAP and MsRNAP, respectively), which were used for in vitro transcription assays and protein-protein interaction studies, were purified by following the method described earlier (9,10).…”

“…(i) Stalled TEC preparation. Ternary elongation complexes (TECs) were generated on a 5= biotinylated T7A1 promoter DNA template using M. tuberculosis RNAP by following the method described earlier (9,18).…”

“…The Gre proteins are the first members of this group of factors to assist RNAP in maintaining transcription accuracy by stimulating the cleavage of aberrant 3= ends of the RNA to resume RNA synthesis (4,5,9,17). Gfh1, DksA, and TraR are structural homologs of Gre factors but do not function like Gre; instead they inhibit the transcription process.…”