Investigation of the Phenotypic Effect of Mutating a Highly-Conserved Cysteine Residue in the RNA Polymerase Beta Prime Subunit of E. Coli RNA Polymerase

All bacteria contain a multi-subunit RNA polymerase (RNAPs) that is essential for gene expression. Because of the centrality of these enzymes in cellular life, the structure and function of the various subunits is intensely studied. The primary sequence of the RNAP β’ subunit contains five cysteine residues that are highly conserved. Four of the cysteines coordinate a zinc atom and form the beta prime subunit zinc binding domain (ZBD). Mutation of any one of the ZBD cysteines is lethal to the cell. However, the role of the fifth residue (C58), which is located upstream of the ZBD cysteines, has not been investigated. In previous work, we cloned a copy of the E. coli rpoC onto a plasmid and changed the cysteine at position 58 to an alanine (C58A). Phenotypic analysis suggested that expression of the mutant subunit from the multi-copy plasmid did not support E. coli growth at high temperatures. In this study, we describe the generation of the C58A mutation in single copy on the chromosome using a chromosomal engineering technique. In addition, we investigated if the mutant subunit affects RNA-mediated transcription antitermination

Regulation of transcription through the secondary channel of RNA polymerase

PhD ThesisThe bacterial transcription factor Gre performs the highly conserved function of stimulating the endonucleolytic activity of RNA polymerase for efficient RNA cleavage, thereby promoting transcription elongation and assisting in transcript fidelity. This dynamic factormediated cleavage has been extensively studied, except for within the unusual Cyanobacteria where, notably, no Gre homologues have yet been identified. To investigate this apparent absence of Gre factor, the RNA polymerase of two cyanobacterial species, Synechococcus elongatus 7942 and Synechocystis sp 6803, were purified and tested for their in vitro transcription activity. Rates of intrinsic RNA cleavage were 20-90x greater for the cyanobacterial polymerases than rates found for Escherichia coli. Further study revealed differences in the bridge helix and trigger loop structural elements of the active site as the possible cause of this increased activity. Mutational analyses indicate a reduced flexibility of these elements which may fix the active site into a closed and more hydrolytically competent state. We propose that in cyanobacteria, the lack of Gre factor is compensated by the unique composition and endogenous ability of the polymerase itself to perform fast and efficient transcript cleavage thus eliminating the need for additional factors. In this work a Gre factor homologue, Gfh1, of Thermus aquaticus is also examined. Gfh1 is known to stimulate transcriptional pausing at a wide variety of pause signals and we present further evidence of preferential activity towards the inhibition of transcription from a pre-translocated stateBBSR

The transcriptional apparatus of Chlamydomonas chloroplasts

The transcriptional apparatus of higher plant chloroplasts is well characterised and consists of a plastid-encoded polymerase (PEP) and a nuclear encoded polymerase (NEP). PEP is dispensable to cell viability. The situation in green algal species, however, is less clear. Chloroplast genes encoding subunits of the PEP have been cloned and sequenced in the green alga Chlamydomonas reinhardtii and preliminary reverse-genetic studies suggest that PEP is essential to cell viability, which is in contrast to the situation in higher plants. To investigate this further a series of gene knockouts were constructed using the chloroplast gene rpoC2, encoding the " subunit of PEP. Results indicate that PEP is essential to C. reinhardtii cell viability. In addition, inhibitors of PEP have been used in an in vivo transcription assay to try to identify a second RNA polymerase activity in C. reinhardtii chloroplasts. In all higher plant and red algal species so far studied the PEP factor is encoded in the nuclear genome. A C. reinhardtii nuclear gene (rpoD) encoding a putative PEP factor has been cloned and partially sequenced. This is the first factor cloned from a green algal species. A transcript of ~2.9 kb was detected for the rpoD gene by northern analysis. Finally, epitope tagging technology was developed for chloroplast and bacterial gene products. The rpoC2 gene of C. reinhardtii was modified to produce a 6x-histidine tagged polypeptide and an attempt was made to purify this polypeptide from C. reinhardtii cells using IMAC. In addition, a 3x haemagglutinin (HA) epitope tag was codon optimised for use in C. reinhardtii chloroplasts and this epitope was used to tag -galactosidase in E. coli. The protein was detected in a western blot using anti-HA monoclonal antibodies. This epitope will prove useful as a tool to tag C. reinhardtii chloroplast proteins

Evolving MRSA : high-level β-lactam resistance in Staphylococcus aureus is associated with RNA Polymerase alterations and fine tuning of gene expression

Most clinical MRSA (methicillin-resistant S. aureus) isolates exhibit low-level β-lactam resistance (oxacillin MIC 2–4 μg/ml) due to the acquisition of a novel penicillin binding protein (PBP2A), encoded by mecA. However, strains can evolve high-level resistance (oxacillin MIC ≥256 μg/ml) by an unknown mechanism. Here we have developed a robust system to explore the basis of the evolution of high-level resistance by inserting mecA into the chromosome of the methicillin-sensitive S. aureus SH1000. Low-level mecA-dependent oxacillin resistance was associated with increased expression of anaerobic respiratory and fermentative genes. High-level resistant derivatives had acquired mutations in either rpoB (RNA polymerase subunit β) or rpoC (RNA polymerase subunit β’) and these mutations were shown to be responsible for the observed resistance phenotype. Analysis of rpoB and rpoC mutants revealed decreased growth rates in the absence of antibiotic, and alterations to, transcription elongation. The rpoB and rpoC mutations resulted in decreased expression to parental levels, of anaerobic respiratory and fermentative genes and specific upregulation of 11 genes including mecA. There was however no direct correlation between resistance and the amount of PBP2A. A mutational analysis of the differentially expressed genes revealed that a member of the S. aureus Type VII secretion system is required for high level resistance. Interestingly, the genomes of two of the high level resistant evolved strains also contained missense mutations in this same locus. Finally, the set of genetically matched strains revealed that high level antibiotic resistance does not incur a significant fitness cost during pathogenesis. Our analysis demonstrates the complex interplay between antibiotic resistance mechanisms and core cell physiology, providing new insight into how such important resistance properties evolve

The involvement of the aspartate triad of the active center in all catalytic activities of multisubunit RNA polymerase

Three conserved aspartate residues in the largest subunit of multisubunit RNA polymerases (RNAPs) coordinate two Mg(2+) ions involved in the catalysis of phosphodiester bond synthesis. A structural model based on the stereochemistry of nucleotidyl transfer reaction as well as recent crystallographic data predict that these Mg(2+) ions should also be involved in the reverse reaction of pyrophosphorolysis as well as in the endo- and exonucleolytic cleavage of the nascent RNA. Here, we check these predictions by constructing point substitutions of each of the three Asp residues in the β′ subunit of Escherichia coli RNAP and testing the mutant enzymes' functions. Using artificially assembled elongation complexes, we demonstrate that substitutions of any of the three aspartates dramatically reduce all known RNAP catalytic activities, supporting the model's predictions that same amino acids participate in all RNAP catalytic reactions. We demonstrate that though substitutions in the DFDGD motif decrease Mg(2+) binding to free RNAP below detection limits, the apparent affinity to Mg(2+) in transcription complexes formed by the mutant and wild-type RNAPs is similar, suggesting that NTP substrates and/or nucleic acids actively contribute to the retention of active center Mg(2+)

Kinetic Characterization of Rifamycin-Resistant M. tuberculosis RNA Polymerases and Novel Therapeutic Approach for Targeting Transcription

Tuberculosis (TB) remains a critical threat to global human health. In 2016, 1.7 million people died from the disease. Rifampin (RMP) remains a key component of the front-line treatment for TB, though resistance has presented challenges for its efficacy. Resistance to RMP (RMPR) primarily occurs through point mutations of its target, RNA polymerase, within the rifamycin resistance determining region (RRDR) in the β-subunit. Three mutations constitute the bulk of RMPR, βD435V, βH445Y, and βS450L, with the latter being most prevalent in clinically resistant isolates. The molecular mechanisms which yield the observed distribution of RMPR mutations in MTB have been speculated upon; however, detailed in vitro studies of Mycobacterium tuberculosis (MTB) RNAP to elucidate those mechanisms have been lacking. This has likely been due, in part, to difficulty in acquiring pure MTB RNAP. To surmount this, an optimized methodology for the expression and purification of highly pure and active MTB RNAP is described. Co-expression of multiple vectors harboring all subunits of the RNAP holoenzyme allows for in vivo assembly of the holo RNAP complex. An optimized purification method was developed to acquire stoichiometric holo RNAP with high activity. In vivo fitness defects have been observed in RMPR mutants of MTB RNAP. These defects have been found to be ameliorated by the presence of secondary mutations in double-psi β-barrel (DPBB) of the RNAP β’-subunit. To identify factors contributing to this fitness defect, several in vitro transcription assays were utilized to probe initiation, elongation, termination and RNA primer hydrolysis with the wild-type and RMPR RNAPs. Secondary, compensatory mutations are predominantly associated with the βS450L mutant, therefore this mutant was also studied in the presence of secondary mutations. We found that the RMPR mutants exhibit significantly poorer termination efficiency relative to wild-type, an important factor for proper gene expression. This may contribute to the relative prevalence of the RMPR mutants observed in MTB clinical isolates. We also found that several mechanistic aspects of transcription of the RifR mutant RNAPs are impacted relative to wild-type, particularly the stability of the open-promoter complex and elongation rate. For the βS450L mutant, these defects are mitigated in the presence of secondary mutations in the DPBB of the β’-subunit, making the intrinsic properties of this mutant similar to those of the wild-type. These data provide insight into the cost of antibiotic resistance to the fitness of the organism and a mechanistic basis for how MTB alleviates fitness defects associated with drug resistance. Drug resistant TB has become pervasive in large part due to a lack of novel therapeutics which act by new mechanisms of action. CarD is a global transcription regulator which acts by stabilizing the open-promoter complex of MTB RNAP and has been shown to be required for MTB viability. This suggests that CarD may be an effective and novel target for therapeutic discovery for the treatment of tuberculosis. A fluorescence polarization assay which monitors the association of MTB RNAP, native rRNA promoter DNA and Bodipy-CarD has been developed, optimized and validated. A high throughput screen has been conducted to identify and characterize small molecule inhibitors which block the CarD•RNAP•DNA interaction. Several preliminary hits have been identified from this screen and initial secondary characterizations have been performed. This project will be the foundation for further investigation of CarD’s potential as a therapeutic target.PHDMedicinal ChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/146094/1/mastefan_1.pd

Interplay between DNA replication, transcription and repair

The Ruv ABC and RecBCD protein complexes together can collapse and repair arrested replication forks. With their help a fork structure can be re-established on which replication can be restarted. ruv and recB mutants are therefore quite sensitive to UV light. Their survival is greatly decreased in the absence of the signalling molecules (p)ppGpp and increased when excess (p)ppGpp is present. (p)ppGpp are the effector molecules of the stringent response, regulating adaptation to starvation and other stressful environmental changes. Absence of (p)ppGpp can be compensated for by mutations in RNA polymerase that are called stringent mutations. Some of those, called rpo *, also - like excess (p)ppGpp - increase the survival of UV irradiated ruv and recB cells. A model proposed by McGlynn and Lloyd (Cell, Vol. 101, pp35-45, March 31, 2000) suggests that this is achieved by modulation of RNA polymerase, which decreases the incidence of replication fork blocks. In this work twenty-seven rpo * mutants were isolated, sequenced and mapped on the 3D structure of Thermus aquatic us RNA polymerase. I have found mutants in the ~ and ~' subunits of RNA polymerase. They lie mostly on the inner surface of the protein, well placed to make contact with the DNA substrate or the RNA product. A large number of rifampicin resistant mutations among rpo* mutations is explained by an overlap between the so-called Rif pocket and the "rpo* pocket". rpo * mutations, like stringent mutations, lead to a decrease in cell size, suppress filamentation and increase viability. For in vitro studies I purified wild type and two mutant RNA polymerases with help of a his-tagged a subunit. The experiments confirmed that rpo* mutant RNA polymerases form less stable open complexes than wild type, just like previously investigated stringent RNA polymerases. In addition I have shown here that (p)ppGpp leads to the destabilisation of RNA polymerase complexes stalled by nucleotide starvation or UV-induced lesions, though there is as yet no indication that rpo * mutations act in the same way

Multiple roles of the RNA polymerase β′ SW2 region in transcription initiation, promoter escape, and RNA elongation

Interactions of RNA polymerase (RNAP) with nucleic acids must be tightly controlled to ensure precise and processive RNA synthesis. The RNAP β′-subunit Switch-2 (SW2) region is part of a protein network that connects the clamp domain with the RNAP body and mediates opening and closing of the active center cleft. SW2 interacts with the template DNA near the RNAP active center and is a target for antibiotics that block DNA melting during initiation. Here, we show that substitutions of a conserved Arg339 residue in the Escherichia coli RNAP SW2 confer diverse effects on transcription that include defects in DNA melting in promoter complexes, decreased stability of RNAP/promoter complexes, increased apparent KM for initiating nucleotide substrates (2- to 13-fold for different substitutions), decreased efficiency of promoter escape, and decreased stability of elongation complexes. We propose that interactions of Arg339 with DNA directly stabilize transcription complexes to promote stable closure of the clamp domain around nucleic acids. During initiation, SW2 may cooperate with the σ3.2 region to stabilize the template DNA strand in the RNAP active site. Together, our data suggest that SW2 may serve as a key regulatory element that affects transcription initiation and RNAP processivity through controlling RNAP/DNA template interactions