Wilma Ross

DksA: A Critical Component of the Transcription Initiation Machinery that Potentiates the Regulation of rRNA Promoters by ppGpp and the Initiating NTP

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.

E.coli Fis protein activates ribosomal RNA transcription in vitro and in vivo.

An upstream activation region (UAR) contributes to the extremely high activity of the Escherichia coli ribosomal RNA promoter, rrnB P1, increasing its activity 20‐ to 30‐fold over that of the same promoter lacking the UAR. We have used DNase footprinting to define three specific sites in the rrnB P1 UAR that bind Fis, a protein identified previously by its role in recombinational enhancer function in other systems. We find that purified Fis activates transcription from promoters containing these sites 10‐ to 20‐fold in vitro at concentrations correlating with the filling of these sites. Three approaches indicate that Fis contributes to the function of the UAR in vivo. First, there is a progressive loss in the activity of rrnB P1‐lacZ fusions as Fis binding sites are deleted. Second, an rrnB P1 promoter with a mutation in a Fis binding site has 5‐fold reduced transcription activity in vivo, dramatically reduced Fis binding in vitro, and shows no Fis dependent transcription activation in vitro. Third, upstream activation is reduced 5‐fold in a Fis‐ strain. We show that rRNA promoters derepress in response to the loss of Fis in vivo in accord with the predictions of the negative feedback model for rRNA regulation. We find that fis is not essential for the function of two control systems known to regulate rRNA, growth rate dependent control and stringent control. On the basis of these results, we propose roles for Fis and the upstream activation system in rRNA synthesis.

Advances in bacterial promoter recognition and its control by factors that do not bind DNA

Early work identified two promoter regions, the −10 and −35 elements, that interact sequence specifically with bacterial RNA polymerase (RNAP). However, we now know that several additional promoter elements contact RNAP and influence transcription initiation. Furthermore, our picture of promoter control has evolved beyond one in which regulation results solely from activators and repressors that bind to DNA sequences near the RNAP binding site: many important transcription factors bind directly to RNAP without binding to DNA. These factors can target promoters by affecting specific kinetic steps on the pathway to open complex formation, thereby regulating RNA output from specific promoters.

UPs and downs in bacterial transcription initiation: the role of the alpha subunit of RNA polymerase in promoter recognition

In recent years, it has become clear that promoter recognition by bacterial RNA polymerase involves interactions not only between core promoter elements and the σ subunit, but also between a DNA element upstream of the core promoter and the α subunit. DNA binding by α can increase transcription dramatically. Here we review the current state of our understanding of the α interaction with DNA during basal transcription initiation (i.e. in the absence of proteins other than RNA polymerase) and activated transcription initiation (i.e. when stimulated by transcription factors).

Domain organization of RNA polymerase α subunit: C-terminal 85 amino acids constitute a domain capable of dimerization and DNA binding

Using limited proteolysis, we show that the Escherichia coli RNA polymerase alpha subunit consists of an N-terminal domain comprised of amino acids 8-241, a C-terminal domain comprised of amino acids 249-329, and an unstructured and/or flexible interdomain linker. We have carried out a detailed structural and functional analysis of an 85 amino acid proteolytic fragment corresponding to the C-terminal domain (alpha CTD-2). Our results establish that alpha CTD-2 has a defined secondary structure (approximately 40% alpha helix, approximately 0% beta sheet). Our results further establish that alpha CTD-2 is a dimer and that alpha CTD-2 exhibits sequence-specific DNA binding activity. Our results suggest a model for the mechanism of involvement of alpha in transcription activation by promoter upstream elements and upstream-binding activator proteins.

Promoter recognition and discrimination by EsigmaS RNA polymerase.

Although more than 30 Escherichia coli promoters utilize the RNA polymerase holoenzyme containing σS (EσS), and it is known that there is some overlap between the promoters recognized by EσS and by the major E. coli holoenzyme (Eσ70), the sequence elements responsible for promoter recognition by EσS are not well understood. To define the DNA sequences recognized best by EσSin vitro, we started with random DNA and enriched for EσS promoter sequences by multiple cycles of binding and selection. Surprisingly, the sequences selected by EσS contained the known consensus elements (−10 and −35 hexamers) for recognition by Eσ70. Using genetic and biochemical approaches, we show that EσS and Eσ70 do not achieve specificity through ‘best fit’ to different consensus promoter hexamers, the way that other forms of holoenzyme limit transcription to discrete sets of promoters. Rather, we suggest that EσS‐specific promoters have sequences that differ significantly from the consensus in at least one of the recognition hexamers, and that promoter discrimination against Eσ70 is achieved, at least in part, by the two enzymes tolerating different deviations from consensus. DNA recognition by EσS versus Eσ70 thus presents an alternative solution to the problem of promoter selectivity.

Transcription activation at Class II CRP-dependent promoters: identification of determinants in the C-terminal domain of the RNA polymerase alpha subunit.

Many transcription factors, including the Escherichia coli cyclic AMP receptor protein (CRP), act by making direct contacts with RNA polymerase. At Class II CRP‐dependent promoters, CRP activates transcription by making two such contacts: (i) an interaction with the RNA polymerase α subunit C‐terminal domain (αCTD) that facilitates initial binding of RNA polymerase to promoter DNA; and (ii) an interaction with the RNA polymerase α subunit N‐terminal domain that facilitates subsequent promoter opening. We have used random mutagenesis and alanine scanning to identify determinants within αCTD for transcription activation at a Class II CRP‐dependent promoter. Our results indicate that Class II CRP‐dependent transcription requires the side chains of residues 265, 271, 285–288 and 317. Residues 285–288 and 317 comprise a discrete 20×10 Å surface on αCTD, and substitutions within this determinant reduce or eliminate cooperative interactions between α subunits and CRP, but do not affect DNA binding by α subunits. We propose that, in the ternary complex of RNA polymerase, CRP and a Class II CRP‐dependent promoter, this determinant in αCTD interacts directly with CRP, and is distinct from and on the opposite face to the proposed determinant for αCTD–CRP interaction in Class I CRP‐dependent transcription.

Allosteric control of Escherichia coli rRNA promoter complexes by DksA

The Escherichia coli DksA protein inserts into the RNA polymerase (RNAP) secondary channel, modifying the transcription initiation complex so that promoters with specific kinetic characteristics are regulated by changes in the concentrations of ppGpp and NTPs. We used footprinting assays to determine the specific kinetic intermediate, RP(I), on which DksA acts. Genetic approaches identified substitutions in the RNAP switch regions, bridge helix, and trigger loop that mimicked, reduced, or enhanced DksA function on rRNA promoters. Our results indicate that DksA binding in the secondary channel of RP(I) disrupts interactions with promoter DNA at least 25 A away, between positions -6 and +6 (the transcription start site is +1). We propose a working model in which the trigger loop and bridge helix transmit effects of DksA to the switch region(s), allosterically affecting switch residues that control clamp opening/closing and/or that interact directly with promoter DNA. DksA thus inhibits the transition to RP(I). Our results illustrate in mechanistic terms how transcription factors can regulate initiation promoter-specifically without interacting directly with DNA.

Contributions of UP Elements and the Transcription Factor FIS to Expression from the Seven rrn P1 Promoters in Escherichia coli

The high activity of the rrnB P1 promoter in Escherichia coli results from a cis-acting DNA sequence, the UP element, and a trans-acting transcription factor, FIS. In this study, we examine the effects of FIS and the UP element at the other six rrn P1 promoters. We find that UP elements are present at all of the rrn P1 promoters, but they make different relative contributions to promoter activity. Similarly, FIS binds upstream of, and activates, all seven rrn P1 promoters but to different extents. The total number of FIS binding sites, as well as their positions relative to the transcription start site, differ at each rrn P1 promoter. Surprisingly, the FIS sites upstream of site I play a much larger role in transcription from most rrn P1 promoters compared to rrnB P1. Our studies indicate that the overall activities of the seven rrn P1 promoters are similar, and the same contributors are responsible for these high activities, but these inputs make different relative contributions and may act through slightly different mechanisms at each promoter. These studies have implications for the control of gene expression of unlinked multigene families.

Molecular anatomy of a transcription activation patch: FIS–RNA polymerase interactions at the Escherichia coli rrnB P1 promoter

FIS, a site‐specific DNA binding and bending protein, is a global regulator of gene expression in Escherichia coli. The ribosomal RNA promoter rrnB P1 is activated 3‐ to 7‐fold in vivo by a FIS dimer that binds a DNA site immediately upstream of the DNA binding site for the C‐terminal domain (CTD) of the α subunit of RNA polymerase (RNAP). In this report, we identify several FIS side chains important specifically for activation of transcription at rrnB P1. These side chains map to positions 68, 71 and 74, in and flanking a surface‐exposed loop adjacent to the helix–turn–helix DNA binding motif of the protein. We also present evidence suggesting that FIS activates transcription at rrnB P1 by interacting with the RNAP αCTD. Our results suggest a model for FIS‐mediated activation of transcription at rrnB P1 that involves interactions between FIS and the RNAP αCTD near the DNA surface. Although FIS and the transcription activator protein CAP have little structural similarity, they both bend DNA, use a similarly disposed activation loop and target the same region of the RNAP αCTD, suggesting that this is a common architecture at bacterial promoters.