High-throughput, fluorescent-aptamer-based measurements of steady-state transcription rates for the Mycobacterium tuberculosis RNA polymerase.

The first step in gene expression is the transcription of DNA sequences into RNA. Regulation at the level of transcription leads to changes in steady-state concentrations of RNA transcripts, affecting the flux of downstream functions and ultimately cellular phenotypes. Changes in transcript levels are routinely followed in cellular contexts via genome-wide sequencing techniques. However, in vitro mechanistic studies of transcription have lagged with respect to throughput. Here, we describe the use of a real-time, fluorescent-aptamer-based method to quantitate steady-state transcription rates of the Mycobacterium tuberculosis RNA polymerase. We present clear controls to show that the assay specifically reports on promoter-dependent, full-length RNA transcription rates that are in good agreement with the kinetics determined by gel-resolved, α-32P NTP incorporation experiments. We illustrate how the time-dependent changes in fluorescence can be used to measure regulatory effects of nucleotide concentrations and identity, RNAP and DNA concentrations, transcription factors, and antibiotics. Our data showcase the ability to easily perform hundreds of parallel steady-state measurements across varying conditions with high precision and reproducibility to facilitate the study of the molecular mechanisms of bacterial transcription.

Molecular dissection of RbpA-mediated regulation of fidaxomicin sensitivity in mycobacteria.

RNA polymerase (RNAP) binding protein A (RbpA) is essential for mycobacterial viability and regulates transcription initiation by increasing the stability of the RNAP-promoter open complex (RPo). RbpA consists of four domains: an N-terminal tail (NTT), a core domain (CD), a basic linker, and a sigma interaction domain. We have previously shown that truncation of the RbpA NTT and CD increases RPo stabilization by RbpA, implying that these domains inhibit this activity of RbpA. Previously published structural studies showed that the NTT and CD are positioned near multiple RNAP-σA holoenzyme functional domains and predict that the RbpA NTT contributes specific amino acids to the binding site of the antibiotic fidaxomicin (Fdx), which inhibits the formation of the RPo complex. Furthermore, deletion of the NTT results in decreased Mycobacterium smegmatis sensitivity to Fdx, but whether this is caused by a loss in Fdx binding is unknown. We generated a panel of rbpA mutants and found that the RbpA NTT residues predicted to directly interact with Fdx are partially responsible for RbpA-dependent Fdx activity in vitro, while multiple additional RbpA domains contribute to Fdx activity in vivo. Specifically, our results suggest that the RPo-stabilizing activity of RbpA decreases Fdx activity in vivo. In support of the association between RPo stability and Fdx activity, we find that another factor that promotes RPo stability in bacteria, CarD, also impacts to Fdx sensitivity. Our findings highlight how RbpA and other factors may influence RNAP dynamics to affect Fdx sensitivity.

Mycobacterium tuberculosis DNA repair helicase UvrD1 is activated by redox-dependent dimerization via a 2B domain cysteine.

Mycobacterium tuberculosis (Mtb) causes tuberculosis and, during infection, is exposed to reactive oxygen species and reactive nitrogen intermediates from the host immune response that can cause DNA damage. UvrD-like proteins are involved in DNA repair and replication and belong to the SF1 family of DNA helicases that use ATP hydrolysis to catalyze DNA unwinding. In Mtb, there are two UvrD-like enzymes, where UvrD1 is most closely related to other family members. Previous studies have suggested that UvrD1 is exclusively monomeric; however, it is well known that Escherichia coli UvrD and other UvrD family members exhibit monomer-dimer equilibria and unwind as dimers in the absence of accessory factors. Here, we reconcile these incongruent studies by showing that Mtb UvrD1 exists in monomer, dimer, and higher-order oligomeric forms, where dimerization is regulated by redox potential. We identify a 2B domain cysteine, conserved in many Actinobacteria, that underlies this effect. We also show that UvrD1 DNA-unwinding activity correlates specifically with the dimer population and is thus titrated directly via increasing positive (i.e., oxidative) redox potential. Consistent with the regulatory role of the 2B domain and the dimerization-based activation of DNA unwinding in UvrD family helicases, these results suggest that UvrD1 is activated under oxidizing conditions when it may be needed to respond to DNA damage during infection.

Domains within RbpA Serve Specific Functional Roles That Regulate the Expression of Distinct Mycobacterial Gene Subsets.

The RNA polymerase (RNAP) binding protein A (RbpA) contributes to the formation of stable RNAP-promoter open complexes (RPo) and is essential for viability in mycobacteria. Four domains have been identified in the RbpA protein, i.e., an N-terminal tail (NTT) that interacts with RNAP ß' and σ subunits, a core domain (CD) that contacts the RNAP ß' subunit, a basic linker (BL) that binds DNA, and a σ-interaction domain (SID) that binds group I and group II σ factors. Limited in vivo studies have been performed in mycobacteria, however, and how individual structural domains of RbpA contribute to RbpA function and mycobacterial gene expression remains mostly unknown. We investigated the roles of the RbpA structural domains in mycobacteria using a panel of rbpA mutants that target individual RbpA domains. The function of each RbpA domain was required for Mycobacterium tuberculosis viability and optimal growth in Mycobacterium smegmatis We determined that the RbpA SID is both necessary and sufficient for RbpA interaction with the RNAP, indicating that the primary functions of the NTT and CD are not solely association with the RNAP. We show that the RbpA BL and SID are required for RPo stabilization in vitro, while the NTT and CD antagonize this activity. Finally, RNA-sequencing analyses suggest that the NTT and CD broadly activate gene expression, whereas the BL and SID activate or repress gene expression in a gene-dependent manner for a subset of mycobacterial genes. Our findings highlight specific outcomes for the activities of the individual functional domains in RbpA.IMPORTANCEMycobacterium tuberculosis is the causative agent of tuberculosis and continues to be the most lethal infectious disease worldwide. Improved molecular understanding of the essential proteins involved in M. tuberculosis transcription, such as RbpA, could provide targets for much needed future therapeutic agents aimed at combatting this pathogen. In this study, we expand our understanding of RbpA by identifying the RbpA structural domains responsible for the interaction of RbpA with the RNAP and the effects of RbpA on transcription initiation and gene expression. These experiments expand our knowledge of RbpA while also broadening our understanding of bacterial transcription in general.

Cooperative stabilization of Mycobacterium tuberculosis rrnAP3 promoter open complexes by RbpA and CarD.

The essential mycobacterial transcriptional regulators RbpA and CarD act to modulate transcription by associating to the initiation complex and increasing the flux of transcript production. Each of these factors interacts directly with the promoter DNA template and with RNA polymerase (RNAP) holoenzyme. We recently reported on the energetics of CarD-mediated open complex stabilization on the Mycobacterium tuberculosis rrnAP3 ribosomal promoter using a stopped-flow fluorescence assay. Here, we apply this approach to RbpA and show that RbpA stabilizes RNAP-promoter open complexes (RPo) via a distinct mechanism from that of CarD. Furthermore, concentration-dependent stopped-flow experiments with both factors reveal positive linkage (cooperativity) between RbpA and CarD with regard to their ability to stabilize RPo The observation of positive linkage between RbpA and CarD demonstrates that the two factors can act on the same transcription initiation complex simultaneously. Lastly, with both factors present, the kinetics of open complex formation is significantly faster than in the presence of either factor alone and approaches that of E. coli RNAP on the same promoter. This work provides a quantitative framework for the molecular mechanisms of these two essential transcription factors and the critical roles they play in the biology and pathology of mycobacteria.

CarD stabilizes mycobacterial open complexes via a two-tiered kinetic mechanism.

CarD is an essential and global transcriptional regulator in mycobacteria. While its biological role is unclear, CarD functions by interacting directly with RNA polymerase (RNAP) holoenzyme promoter complexes. Here, using a fluorescent reporter of open complex, we quantitate RPo formation in real time and show that Mycobacterium tuberculosis CarD has a dramatic effect on the energetics of RNAP bound complexes on the M. tuberculosis rrnAP3 ribosomal RNA promoter. The data reveal that Mycobacterium bovis RNAP exhibits an unstable RPo that is stabilized by CarD and suggest that CarD uses a two-tiered, concentration-dependent mechanism by associating with open and closed complexes with different affinities. Specifically, the kinetics of open-complex formation can be explained by a model where, at saturating concentrations of CarD, the rate of bubble collapse is slowed and the rate of opening is accelerated. The kinetics and open-complex stabilities of CarD mutants further clarify the roles played by the key residues W85, K90 and R25 previously shown to affect CarD-dependent gene regulation in vivo. In contrast to M. bovis RNAP, Escherichia coli RNAP efficiently forms RPo on rrnAP3, suggesting an important difference between the polymerases themselves and highlighting how transcriptional machinery can vary across bacterial genera.

The YEATS domain of Taf14 in Saccharomyces cerevisiae has a negative impact on cell growth.

The role of a highly conserved YEATS protein motif is explored in the context of the Taf14 protein of Saccharomyces cerevisiae. In S. cerevisiae, Taf14 is a protein physically associated with many critical multisubunit complexes including the general transcription factors TFIID and TFIIF, the chromatin remodeling complexes SWI/SNF, Ino80 and RSC, Mediator and the histone modification enzyme NuA3. Taf14 is a member of the YEATS superfamily, conserved from bacteria to eukaryotes and thought to have a transcription stimulatory activity. However, besides its ubiquitous presence and its links with transcription, little is known about Taf14's role in the nucleus. We use structure-function and mutational analysis to study the function of Taf14 and its well conserved N-terminal YEATS domain. We show here that the YEATS domain is not necessary for Taf14's association with these transcription and chromatin remodeling complexes, and that its presence in these complexes is dependent only on its C-terminal domain. Our results also indicate that Taf14's YEATS domain is not necessary for complementing the synthetic lethality between TAF14 and the general transcription factor TFIIS (encoded by DST1). Furthermore, we present evidence that the YEATS domain of Taf14 has a negative impact on cell growth: its absence enables cells to grow better than wild-type cells under stress conditions, like the microtubule destabilizing drug benomyl. Moreover, cells expressing solely the YEATS domain grow worser than cells expressing any other Taf14 construct tested, including the deletion mutant. Thus, this highly conserved domain should be considered part of a negative regulatory loop in cell growth.

The Pseudomonas putida Crc global regulator is an RNA binding protein that inhibits translation of the AlkS transcriptional regulator.

The Crc protein is a global regulator that controls the hierarchical assimilation of carbon sources in Pseudomonads by inhibiting expression of several catabolic pathways. Crc does not bind DNA and its mechanism of action has remained elusive. Among other genes, Crc inhibits expression of alkS, the transcriptional activator of the Pseudomonas putida OCT plasmid alkane degradation pathway. AlkS activates expression of its own gene. In the presence of saturating AlkS levels, translational fusions of alkS to the lacZ reporter gene were responsive to Crc, but transcriptional fusions were not. In translational fusions, the first 33 nt of alkS mRNA, which includes up to position +3 relative to the translation start site, were sufficient to confer an efficient response to Crc. In vitro, purified Crc could bind specifically to an alkS mRNA fragment spanning positions +1 to +43, comprising the translation initiation region. We have previously shown that Crc has little effect on the stability of alkS mRNA. We conclude that Crc modulates AlkS levels by binding to the translation initiation region of alkS mRNA, thereby inhibiting translation. Because AlkS is an unstable protein present in limiting amounts, reducing its levels leads to decreased expression of all genes in the pathway.

Levels and activity of the Pseudomonas putida global regulatory protein Crc vary according to growth conditions.

The global regulatory protein Crc is involved in the repression of several catabolic pathways for sugars, hydrocarbons, and nitrogenated and aromatic compounds in Pseudomonas putida and Pseudomonas aeruginosa when other preferred carbon sources are present in the culture medium (catabolite repression), therefore modulating carbon metabolism. We have analyzed whether the levels or the activity of Crc is regulated. Crc activity was followed by its ability to inhibit the induction by alkanes of the P. putida OCT plasmid alkane degradation pathway when cells grow in a complete medium, where the effect of Crc is very strong. The abundance of crc transcripts and the amounts of Crc protein were higher under repressing conditions than under nonrepressing conditions. The presence of crc on a high-copy-number plasmid considerably increased Crc levels, but this impaired its ability to inhibit the alkane degradation pathway. Crc shows similarity to a family of nucleases that have highly conserved residues at their catalytic sites. Mutation of the corresponding residues in Crc (Asp220 and His246) led to proteins that can inhibit induction of the alkane degradation pathway when present at normal or elevated levels in the cell. Repression by these mutant proteins occurred only under repressing conditions. These results suggest that both the amounts and the activity of Crc are modulated and support previous proposals that Crc may form part of a signal transduction pathway. Furthermore, the activity of the mutant proteins suggests that Crc is not a nuclease.

Inactivation of cytochrome o ubiquinol oxidase relieves catabolic repression of the Pseudomonas putida GPo1 alkane degradation pathway.

Expression of the alkane degradation pathway encoded by the OCT plasmid of Pseudomonas putida GPo1 is regulated by two control systems. One relies on the transcriptional regulator AlkS, which activates expression of the pathway in the presence of alkanes. The other, which is a dominant global regulation control, represses the expression of the pathway genes when a preferred carbon source is present in the growth medium in addition to alkanes. This catabolite repression control occurs through a poorly characterized mechanism that ultimately regulates transcription from the two AlkS-activated promoters of the pathway. To identify the factors involved, a screening method was developed to isolate mutants without this control. Several isolates were obtained, all of which contained mutations that mapped to genes encoding cytochrome o ubiquinol oxidase, the main terminal oxidase of the electron transport chain under highly aerobic conditions. Elimination of this terminal oxidase led to a decrease in the catabolic repression observed both in rich Luria-Bertani medium and in a defined medium containing lactate or succinate as the carbon source. This suggests that catabolic repression could monitor the physiological or metabolic status by using information from the electron transport chain or from the redox state of the cell. Since inactivation of the crc gene also reduces catabolic repression in rich medium (although not that observed in a defined medium), a strain was generated lacking both the Crc function and the cytochrome o terminal oxidase. The two mutations had an additive effect in relieving catabolic repression in rich medium. This suggests that crc and cyo belong to different regulation pathways, both contributing to catabolic repression.