Deletion of the Gene rpoZ , Encoding the ω Subunit of RNA Polymerase, in Mycobacterium smegmatis Results in Fragmentation of the β′ Subunit in the Enzyme Assembly

Abstract

A deletion mutation in the gene rpoZ of Mycobacterium smegmatis causes reduced growth rate and a change in colony morphology. During purification of RNA polymerase from the mutant strain, the ␤ subunit undergoes fragmentation but the fragments remain associated with the enzyme and maintain it in an active state until the whole destabilized assembly breaks down in the final step of purification. Complementation of the mutant strain with an integrated copy of the wild-type rpoZ brings back the wild-type colony morphology and improves the growth rate and activity of the enzyme, and the integrity of the ␤ subunit remains unaffected.DNA-dependent RNA polymerase (RNAP) is the central enzyme involved in gene expression and also constitutes a major target for genetic regulation (7,8,31). The bacterial RNAP core enzyme consists of four subunits: alpha (␣), beta (␤), betaЈ (␤Ј) and omega () (21, 36). The subunit is the least well studied among all the subunits, though the subunit encoded by the rpoZ gene was proposed to be an integral part of the core RNAP several years ago (5, 10). In Escherichia coli is not found to be necessary for survival of the bacterium under laboratory conditions (12). At the same time homologues are present in the sequenced genomes of free-living bacteria, suggesting an important and conserved role for the protein (23).It was identified in our laboratory that is required for the restoration of denatured core RNAP to its functionally active form (25). Further, we showed that the enzyme purified from an E. coli strain lacking recruits large amounts of GroEL (26) and removal of GroEL results in a completely inactive core RNAP which lacks the ability to even associate with 70 (24). Subsequently, it was demonstrated that binds to the ␤Ј subunit and promotes RNAP assembly by facilitating the association of ␤Ј with the previous step of the assembly, ␣ 2 ␤ (13).The X-ray crystal structure of Thermus aquaticus RNAP determined at 3.3 Å resolution (36) and subsequent analysis of the -␤Ј interface by Minakhin et al. (23) identified the conserved regions of ␤Ј which binds with in a manner that reduces the configurational entropy of ␤Ј and facilitates its interaction with the ␣ 2 ␤ subassembly. Further experiments in our laboratory showed that the C-terminal tail of the subunit is constrained in the presence of ␤Ј (14).In spite of this well established evidence, a number of recent observations about the subunit in different organisms warrant suspecting functional roles for this protein which are not clearly elucidated yet. In Streptomyces kasugaensis, it was observed that a mutation in the gene encoding the subunit resulted in characteristic pleiotropic effects (19). The Kranz laboratory recently demonstrated that presence of was a prerequisite to obtain an active in vitro assembly of Rhodobacter capsulatus RNAP (30). In a completely different scenario, Periago et al. (29) demonstrated induction of YloH, the subunit of Bacillus cereus, by heat stress, suggesting a role for the subunit in stress adaptation of...

Association of ω with the C-Terminal Region of the β′ Subunit Is Essential for Assembly of RNA Polymerase in Mycobacterium tuberculosis

“…In this study, we provide clear evidence (in vivo and in vitro assembly) that, in contrast to E. coli, M. tuberculosis core could not be well assembled and almost lost its function in the absence of . In addition, a previous study has shown that deletion of the rpoZ gene in M. smegmatis resulted in proteolytic cleavage of RNAP ␤= subunit and further affected the function of RNAP in vivo (18). All these data indicate that mycobacterial plays more important roles than that of E. coli .…”

“…First, Mycobacterium leprae, which has lost many genes during the process of reductive evolution, still preserves the subunit (8,17). Second, deletion of rpoZ, a gene encodes the subunit, significantly decreased the growth rate of Mycobacterium smegmatis and resulted in proteolytic cleavage of the RNAP ␤= subunit (18). Third, rpoZ has been predicted to be an essential gene in M. tuberculosis by transposon mutagenesis screening (19,20).…”

Understanding butanol tolerance and assimilation in Pseudomonas putida BIRD‐1: an integrated omics approach

“…An rpo Z mutant [ribonucleic acid (RNA) polymerase accessory protein] exhibited strongly impaired growth in the presence of the stressor and was unable to assimilate butanol as sole carbon source. This is likely due to the role of the RpoZ protein in RNA polymerase stability (Mukherjee et al ., ; Mathew et al ., ) along with potential polar effects on the gene encoding SpoT, which influences the cellular content of ppGpp alarmone (Gentry and Cashel, ). In addition, a single mutant in glutamyl‐Q tRNA (Asp) synthetase ( gluQ , translation) was defective in butanol assimilation and tolerance due to its involvement in general metabolism.…”

Proteomics and mass spectrometric studies reveal planktonic growth of Mycobacterium smegmatis in biofilm cultures in the absence of rpoZ

“…Next, we subjected the strain mcdrzco for proteome analysis in the similar way (not shown , presented as a supplementary material). mcdrzco was generated by complimenting rpoZ in mcdrz [30], where rpoZ was integrated back to the genome presumably at a different location from the original. The 2D profile showed similar pattern as that of wild type (with respect to spots 1, 2 and 3).…”

“…, the smallest subunit, has been shown to be an integral part of the assembly which helps to fold the ␤ subunit and protect it physically [29,30]. We have reported earlier that the rpoZ-deleted mc 2 155 strain (mcdrz) displays various pleiotropic phenotypes like altered colony morphology, minimal sliding motility, defective biofilm formation and inability to produce extracellular matrix [30,31].…”