The context-dependent influence of promoter sequence motifs on transcription initiation kinetics and regulation

The fitness of an individual bacterial cell is highly dependent upon the temporal tuning of gene expression levels when subjected to different environmental cues. Kinetic regulation of transcription initiation is a key step in modulating the levels of transcribed genes to promote bacterial survival. The initiation phase encompasses the binding of RNA polymerase (RNAP) to promoter DNA and a series of coupled protein-DNA conformational changes prior to entry into processive elongation. The time required to complete the initiation phase can vary by orders of magnitude and is ultimately dictated by the DNA sequence of the promoter. In this review, we aim to provide the required background to understand how promoter sequence motifs may affect initiation kinetics during promoter recognition and binding, subsequent conformational changes which lead to DNA opening around the transcription start site, and promoter escape. By calculating the steady-state flux of RNA production as a function of these effects, we illustrate that the presence/absence of a consensus promoter motif cannot be used in isolation to make conclusions regarding promoter strength. Instead, the entire series of linked, sequence-dependent structural transitions must be considered holistically. Finally, we describe how individual transcription factors take advantage of the broad distribution of sequence-dependent basal kinetics to either increase or decrease RNA flux

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

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 RP(o) 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 RP(o) 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 RP(o) on rrnAP3, suggesting an important difference between the polymerases themselves and highlighting how transcriptional machinery can vary across bacterial genera

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 (R

Effects of increasing the affinity of CarD for RNA polymerase on Mycobacterium tuberculosis growth, rRNA transcription, and virulence

CarD is an essential RNA polymerase (RNAP) interacting protein in Mycobacterium tuberculosis that stimulates formation of RNAP-promoter open complexes. CarD plays a complex role in M. tuberculosis growth and virulence that is not fully understood. Therefore, to gain further insight into the role of CarD in M. tuberculosis growth and virulence, we determined the effect of increasing the affinity of CarD for RNAP. Using site-directed mutagenesis guided by crystal structures of CarD bound to RNAP, we identified amino acid substitutions that increase the affinity of CarD for RNAP. Using these substitutions, we show that increasing the affinity of CarD for RNAP increases the stability of the CarD protein in M. tuberculosis. In addition, we show that increasing the affinity of CarD for RNAP increases the growth rate in M. tuberculosis without affecting 16S rRNA levels. We further show that increasing the affinity of CarD for RNAP reduces M. tuberculosis virulence in a mouse model of infection despite the improved growth rate in vitro. Our findings suggest that the CarD-RNAP interaction protects CarD from proteolytic degradation in M. tuberculosis, establish that growth rate and rRNA levels can be uncoupled in M. tuberculosis and demonstrate that the strength of the CarD-RNAP interaction has been finely tuned to optimize virulence. IMPORTANCE Mycobacterium tuberculosis, the causative agent of tuberculosis, remains a major global health problem. In order to develop new strategies to battle this pathogen, we must gain a better understanding of the molecular processes involved in its survival and pathogenesis. We have previously identified CarD as an essential transcriptional regulator in mycobacteria. In this study, we detail the effects of increasing the affinity of CarD for RNAP on transcriptional regulation, CarD protein stability, and virulence. These studies expand our understanding of the global transcription regulator CarD, provide insight into how CarD activity is regulated, and broaden our understanding of prokaryotic transcription

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 (R

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 (RP(o)) 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 RP(o). 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

Structural and computational studies of two oligonucleotide modifying enzymes: I-PpoI and T4 polynucleotide kinase

Thesis (Ph. D.)--University of Washington, 2002Endonucleases are a large class of enzymes that catalyze the formation of site-specific double strand breaks in DNA. Homing endonucleases are expressed from open reading frames within intervening sequences (introns or inteins) and promote the lateral transfer of their host intron to a cognate intronless allele. A novel mechanism of DNA endonucleolytic cleavage, exhibited by the homing endonuclease I-Ppol and characterized by a histidine general base, has been visualized at high resolution by intermediate trapping and studied computationally. The structures of three separate catalytic species were determined by X-ray crystallography and are presented here. The nudged elastic band method was used to calculate the minimum energy path for the catalyzed reaction and the predicted rate is compared to experiment. In addition, the structure of I-Ppol bound to its DNA target site shows that bound DNA is severely bent, resulting in significant deformations of the minor and major grooves near the scissile phosphates. To study the role of conformational changes within the protein catalyst and the DNA substrate, the structures of both the apo enzyme and the L116A mutant protein/DNA complex with severely decreased binding affinity were determined. The wild-type protein displays a rigid-body rotation of its individual subunits upon DNA binding. Homing site DNA is not detectably bent in the absence of protein, but is sharply bent in both the WT and L116A complexes, indicating that binding involves a large distortion of the DNA and a smaller change in protein conformation. The critical function of leucine 116 is also described.T4 phage polynucleotide kinase (PNK) was identified over 35 years ago and has become a staple reagent for molecular biologists. The enzyme displays 5'-hydroxyl kinase, 3'-phosphatase, and 2',3'-cyclic phosphodiesterase activities against a wide range of substrates. These activities modify the ends of a nicked tRNA generated by a bacterial response to infection and facilitate their repair by T4 RNA ligase. DNA repair enzymes that share conserved motifs with PNK have been identified in eukaryotes. PNK contains two functionally distinct structural domains and forms a homotetramer. The C-terminal phosphatase domain is homologous to the L-2 haloacid dehalogenase family and the N-terminal kinase domain is homologous to adenylate kinase. The active sites have been characterized through structural homology analyses and visualization of bound substrate