Brian J Paul

University of Wisconsin, Madison, Madison, MS, United States

Are you Brian J Paul?

Claim your profile

Publications (6)76.37 Total impact

  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: DksA is a critical transcription factor in Escherichia coli that binds to RNA polymerase and potentiates control of rRNA promoters and certain amino acid promoters. Given the kinetic similarities between rRNA promoters and the fis promoter (Pfis), we investigated the possibility that DksA might also control transcription from Pfis. We show that the absence of dksA extends transcription from Pfis well into the late logarithmic and stationary growth phases, demonstrating the importance of DksA for growth phase-dependent regulation of fis. We also show that transcription from Pfis increases with steady-state growth rate and that dksA is absolutely required for this regulation. In addition, both DksA and ppGpp are required for inhibition of Pfis promoter activity following amino acid starvation, and these factors act directly and synergistically to negatively control Pfis transcription in vitro. DksA decreases the half-life of the intrinsically short-lived fis promoter-RNA polymerase complex and increases its sensitivity to the concentration of CTP, the predominant initiating nucleotide triphosphate for this promoter. This work extends our understanding of the multiple factors controlling fis expression and demonstrates the generality of the DksA requirement for regulation of kinetically similar promoters.
    Journal of Bacteriology 09/2006; 188(16):5775-82. · 2.69 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Amino acid starvation in Escherichia coli results in a spectrum of changes in gene expression, including inhibition of rRNA and tRNA promoters and activation of certain promoters for amino acid biosynthesis and transport. The unusual nucleotide ppGpp plays an important role in both negative and positive regulation. Previously, we and others suggested that positive effects of ppGpp might be indirect, resulting from the inhibition of rRNA transcription and, thus, liberation of RNA polymerase for binding to other promoters. Recently, we showed that DksA binds to RNA polymerase and greatly enhances direct effects of ppGpp on the negative control of rRNA promoters. This conclusion prompted us to reevaluate whether ppGpp might also have a direct role in positive control. We show here that ppGpp greatly increases the rate of transcription initiation from amino acid promoters in a purified system but only when DksA is present. Activation occurs by stimulation of the rate of an isomerization step on the pathway to open complex formation. Consistent with the model that ppGpp/DksA stimulates amino acid promoters both directly and indirectly in vivo, cells lacking dksA fail to activate transcription from the hisG promoter after amino acid starvation. Our results illustrate how transcription factors can positively regulate transcription initiation without binding DNA, demonstrate that dksA directly affects promoters in addition to those for rRNA, and suggest that some of the pleiotropic effects previously associated with dksA might be ascribable to direct effects of dksA on promoters involved in a wide variety of cellular functions.
    Proceedings of the National Academy of Sciences 06/2005; 102(22):7823-8. · 9.81 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Ribosomal RNA (rRNA) transcription is regulated primarily at the level of initiation from rRNA promoters. The unusual kinetic properties of these promoters result in their specific regulation by two small molecule signals, ppGpp and the initiating NTP, that bind to RNA polymerase (RNAP) at all promoters. We show here that DksA, a protein previously unsuspected as a transcription factor, is absolutely required for rRNA regulation. In deltadksA mutants, rRNA promoters are unresponsive to changes in amino acid availability, growth rate, or growth phase. In vitro, DksA binds to RNAP, reduces open complex lifetime, inhibits rRNA promoter activity, and amplifies effects of ppGpp and the initiating NTP on rRNA transcription, explaining the dksA requirement in vivo. These results expand our molecular understanding of rRNA transcription regulation, may explain previously described pleiotropic effects of dksA, and illustrate how transcription factors that do not bind DNA can nevertheless potentiate RNAP for regulation.
    Cell 09/2004; 118(3):311-22. · 33.12 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Ribosomal RNA transcription is the rate-limiting step in ribosome synthesis in bacteria and has been investigated intensely for over half a century. Multiple mechanisms ensure that rRNA synthesis rates are appropriate for the cell's particular growth condition. Recently, important advances have been made in our understanding of rRNA transcription initiation in Escherichia coli. These include (a) a model at the atomic level of the network of protein-DNA and protein-protein interactions that recruit RNA polymerase to rRNA promoters, accounting for their extraordinary strength; (b) discovery of the nonredundant roles of two small molecule effectors, ppGpp and the initiating NTP, in regulation of rRNA transcription initiation; and (c) identification of a new component of the transcription machinery, DksA, that is absolutely required for regulation of rRNA promoter activity. Together, these advances provide clues important for our molecular understanding not only of rRNA transcription, but also of transcription in general.
    Annual Review of Genetics 02/2004; 38:749-70. · 18.12 Impact Factor
  • Article: DksA
    [Show abstract] [Hide abstract]
    ABSTRACT: Ribosomal RNA (rRNA) transcription is regulated primarily at the level of initiation from rRNA promoters. The unusual kinetic properties of these promoters result in their specific regulation by two small molecule signals, ppGpp and the initiating NTP, that bind to RNA polymerase (RNAP) at all promoters. We show here that DksA, a protein previously unsuspected as a transcription factor, is absolutely required for rRNA regulation. In ΔdksA mutants, rRNA promoters are unresponsive to changes in amino acid availability, growth rate, or growth phase. In vitro, DksA binds to RNAP, reduces open complex lifetime, inhibits rRNA promoter activity, and amplifies effects of ppGpp and the initiating NTP on rRNA transcription, explaining the dksA requirement in vivo. These results expand our molecular understanding of rRNA transcription regulation, may explain previously described pleiotropic effects of dksA, and illustrate how transcription factors that do not bind DNA can nevertheless potentiate RNAP for regulation.
    Cell. 01/2004; 118(3):311-322.
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The C-terminal domain of the Escherichia coli RNA polymerase (RNAP) alpha subunit (alphaCTD) stimulates transcription initiation by interacting with upstream (UP) element DNA and a variety of transcription activators. Here we identify specific substitutions in region 4.2 of sigma 70 (sigma(70)) and in alphaCTD that decrease transcription initiation from promoters containing some, but not all, UP elements. This decrease in transcription derives from a decrease in the initial equilibrium constant for RNAP binding (K(B)). The open complexes formed by the mutant and wild-type RNAPs differ in DNAse I sensitivity at the junction of the alphaCTD and sigma DNA binding sites, correlating with the differences in transcription. A model of the DNA-alphaCTD-sigma region 4.2 ternary complex, constructed from the previously determined X-ray structures of the Thermus aquaticus sigma region 4.2-DNA complex and the E. coli alphaCTD-DNA complex, indicates that the residues identified by mutation in sigma region 4.2 and in alphaCTD are in very close proximity. Our results strongly suggest that alphaCTD, when bound to an UP element proximal subsite, contacts the RNAP sigma(70) subunit, increasing transcription. Previous data from the literature suggest that this same sigma-alphaCTD interaction also plays a role in transcription factor-mediated activation.
    Genes & Development 06/2003; 17(10):1293-307. · 12.64 Impact Factor