An Insertion in the Catalytic Trigger Loop Gates the Secondary Channel of RNA Polymerase

https://doi.org/10.1016/j.jmb.2012.11.008Get rights and content

Abstract

Escherichia coli DksA and GreB bind to RNA polymerase (RNAP), reaching inside the secondary channel, with similar affinities but have different cellular functions. DksA destabilizes promoter complexes whereas GreB facilitates RNA cleavage in arrested elongation complexes (ECs). Although the less abundant GreB may not interfere with DksA regulation during initiation, reports that DksA acts during elongation and termination suggest that it may exclude GreB from arrested complexes, potentially triggering genome instability. Here, we show that GreB does not compete with DksA during termination whereas DksA, even when present in several hundredfold molar excess, does not inhibit GreB-mediated cleavage of the nascent RNA. Our findings that DksA does not bind to backtracked or active ECs provide an explanation for the lack of DksA activity on most ECs that we reported previously, raising a question of what makes a transcription complex susceptible to DksA. Structural modeling suggests that i6, an insertion in the catalytic trigger loop, hinders DksA access into the channel, restricting DksA action to a subset of transcription complexes. In support of this hypothesis, we demonstrate that deletion of i6 permits DksA binding to ECs and that the distribution of DksA and i6 in bacterial genomes is strongly concordant. We hypothesize that DksA binds to transcription complexes in which i6 becomes mobile, for example, as a consequence of weakened RNAP interactions with the downstream duplex DNA.

Graphical Abstract

Highlights

► DksA and GreB bind to the same site on free RNAP. ► DksA does not bind to ECs and cannot compete with GreB. ► The i6 domain prevents DksA binding to transcription complexes. ► All bacterial genomes that have DksA also have i6. ► DksA may target transcription complexes in which i6 becomes mobile.

Introduction

In multi-subunit RNA polymerases (RNAPs), the active site is accessible from the outside via the secondary channel (SC; also called the pore in pol II). Regulatory proteins that bind within this channel control transcription through altering properties of RNAP. These proteins consist of an extended domain, which binds within the SC, and a globular domain, which binds to the RNAP surface outside of the SC. The sequences and even structures of these proteins can be very different and, thus, are their effects on transcription. In Escherichia coli, five different SC regulators, DksA, GreA, GreB, Rnk and TraR,[1], [2], [3], [4], [5] have been characterized and other candidates are suggested by genome analysis.

E. coli GreA, GreB and DksA share the basic two-domain architecture, an extended coiled-coil (CC) domain and a globular domain (Fig. 1a), but play very different roles in the cell. DksA functions predominantly during initiation to tune rRNA synthesis to cellular cues.6 Gre factors rescue elongation complexes (ECs) that become arrested when RNAP makes an error or runs into a roadblock.7 However, the effects of GreB and DksA are not limited to a single step in the transcription cycle; recent studies demonstrate that both factors affect initiation and elongation[8], [9] and could play partially overlapping roles in DNA repair.[10], [11], [12] GreB and DksA are thought to interact with the same RNAP region, the β′ rim helices (RH) domain,[5], [8] and bind to free RNAP with similar affinities9 but do not interact with nucleic acids. It is therefore unclear how they recognize their cellular targets and avoid competition with each other.

GreB and DksA are present at constant levels in the cell, but DksA is 10 times more abundant.9 Thus, GreB would not be expected to interfere with DksA during transcription initiation but, when overexpressed, can substitute for some (negative control of rRNA synthesis) but not other (activation of amino acid biosynthetic genes) activities of DksA.9 Conversely, since DksA affects RNA chain elongation but lacks the ability of GreB to enhance the nascent RNA cleavage,8 DksA could, in principle, inhibit GreB function by blocking GreB binding to arrested ECs. This hypothetical competition would be avoided if DksA and GreB recognized different subsets of ECs; indeed, GreB action appears to be restricted to ECs in which the β′ trigger loop (TL) is unfolded.7

We recently reported that E. coli DksA, and especially its hyperactive variant DksAN88I that increases affinity for free RNAP,13 decreased the rate of elongation and increased termination.8 However, we could not detect any DksA effect on isolated ECs, leaving an identity of its target unknown. Interestingly, DksA activity during elongation was strongly augmented by a deletion of a species-specific insertion in the TL (called i6 or SI3; E. coli β′ residues 943–1130), in sharp contrast to the resistance of the Δi6 RNAP to GreB-mediated cleavage.14 We hypothesized that DksA and GreB bind to different subsets of ECs and that i6 plays a key role in this discrimination, either directly, by modulating the transcription factor binding, or indirectly, through a coupled conformational change in the TL.

Here, we present evidence for a direct effect of i6 on DksA recruitment. Structural modeling suggests a mechanism where i6 physically hinders DksA access into the channel, and we show that the deletion of i6 increases DksA affinity for the ECs but not for core RNAP. Consistent with the key role of i6 in selective DksA recruitment, we find that i6 is present in every bacterial genome that has DksA. Based on these observations, we hypothesize that the DksA effect on elongation and termination reported by us previously8 is mediated through targeting of a transient intermediate in which the i6 position is altered, for example, due to changes in RNAP/DNA interactions or conformational transitions of the TL.

Section snippets

DksA and GreB bind to the same target on RNAP

E. coli DksA and GreB have no sequence homology but share strikingly similar architecture (Fig. 1a) and interact with the same sites on RNAP. Structurally similar CC domains bind inside the SC, positioning the two acidic residues near the RNAP active site; •OH radicals generated by the Fe2 + ion bound in place of the catalytic Mg2 + ion induce cleavage at the tip of the CC of GreB and DksA (Fig. 1b). Structurally different globular domains are thought to interact with the β′ RH domain that lies

Discussion

Here, we show that although DksA and GreB compete for binding to free RNAP, they do not interfere with each other's activities during elongation. We suggest that these and other SC factors bind to different conformations of the EC, which are in turn dictated by TL and adjacent β′ domains. Most importantly, we show that the i6 insertion in the β′ subunit controls DksA binding to the EC.

Reagents

Oligonucleotides were obtained from Integrated DNA Technologies (Coralville, IA); NTPs, from GE Healthcare (Piscataway, NJ); 32P-NTPs, from Perkin Elmer (Waltham, MA); restriction and modification enzymes, from NEB (Ipswich, MA); PCR reagents, from Roche (Indianapolis, IN); other chemicals, from Sigma (St. Louis, MO) and Fisher (Pittsburgh, PA). Plasmid DNAs and PCR products were purified using spin kits from Qiagen (Valencia, CA) and Promega (Madison, WI). All plasmids are listed in Table 1.

Proteins

Acknowledgements

We thank Seth Darst for providing a model of E. coli GreB bound to EC and Georgy Belogurov and the members of the Gourse laboratory for stimulating discussions. This work was supported by the National Science Foundation (MCB-0949569; I.A.) and by the Intramural Research Program of the National Library of Medicine at National Institutes of Health (Y.I.W.).

References (42)

  • E. James et al.

    Structural and mechanistic basis for the inhibition of Escherichia coli RNA polymerase by T7 Gp2

    Mol. Cell

    (2012)
  • I. Artsimovitch et al.

    Co-overexpression of Escherichia coli RNA polymerase subunits allows isolation and analysis of mutant enzymes lacking lineage-specific sequence insertions

    J. Biol. Chem.

    (2003)
  • J. Ederth et al.

    The downstream DNA jaw of bacterial RNA polymerase facilitates both transcriptional initiation and pausing

    J. Biol. Chem.

    (2002)
  • I. Toulokhonov et al.

    A central role of the RNA polymerase trigger loop in active-site rearrangement during transcriptional pausing

    Mol. Cell

    (2007)
  • I. Artsimovitch et al.

    Tagetitoxin inhibits RNA polymerase through trapping of the trigger loop

    J. Biol. Chem.

    (2011)
  • O. Laptenko et al.

    Biochemical assays of Gre factors of Thermus thermophilus

    Methods Enzymol.

    (2003)
  • G.A. Belogurov et al.

    Structural basis for converting a general transcription factor into an operon-specific virulence regulator

    Mol. Cell

    (2007)
  • M.D. Blankschien et al.

    TraR, a homolog of a RNAP secondary channel interactor, modulates transcription

    PLoS Genet.

    (2009)
  • O. Laptenko et al.

    Transcript cleavage factors GreA and GreB act as transient catalytic components of RNA polymerase

    EMBO J.

    (2003)
  • M.N. Vassylyeva et al.

    The carboxy-terminal coiled-coil of the RNA polymerase β′-subunit is the main binding site for Gre factors

    EMBO Rep.

    (2007)
  • S.P. Haugen et al.

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

    Nat. Rev., Microbiol.

    (2008)
  • Cited by (32)

    • Role of the trigger loop in translesion RNA synthesis by bacterial RNA polymerase

      2020, Journal of Biological Chemistry
      Citation Excerpt :

      It was found that the addition of DksA/ppGpp indeed decreased translesion transcription by the ΔSI3 RNAP; in particular, the efficiency of overall RNA extension after 30 min was decreased from ∼60% to ∼30% (Fig. 8, A and B; the fraction of nonextended RNA is shown in blue). Similarly, DksA alone could inhibit translesion synthesis by the ΔSI3 RNAP but not WT RNAP (Fig. S6), suggesting that the SI3 domain indeed “gates” the secondary RNAP channel against binding of DksA (60). The inhibitory effect of DksA/ppGpp on RNA extension was partially suppressed by the G1136M(ΔSI3) substitution (Fig. 8B and Fig. S6, bottom panels).

    • The Mechanisms of Substrate Selection, Catalysis, and Translocation by the Elongating RNA Polymerase

      2019, Journal of Molecular Biology
      Citation Excerpt :

      Deletion of rpb9 is synthetically lethal with rpb1-E1103G [231], a substitution in the TL that stabilizes its closed conformation [44]. The β′ rim helices-bound GreB and DksA are located near the SI3 domain [233], and their functions are strongly dependent on SI3 [42,234]. This insertion is absent in most bacteria and in eukaryotes, but Rpb9 position on the TEC is similar to that of SI3 and Rpb9 has been proposed to modulate TL folding similarly to SI3 [231].

    • Allosteric Effector ppGpp Potentiates the Inhibition of Transcript Initiation by DksA

      2018, Molecular Cell
      Citation Excerpt :

      β′i6 was not resolved in any of the structures determined in this study, indicating that it remains flexible. This observation is consistent with the proposal that DksA only binds to forms of RNAP in which β′i6 is mobile (Furman et al., 2013). The CC tip, which is required for DksA function (Lee et al., 2012), inserts into the secondary channel and comes within ∼16 Å of the catalytic Mg2+ coordinated at the active site (Figure 1B).

    • PpGpp Binding to a Site at the RNAP-DksA Interface Accounts for Its Dramatic Effects on Transcription Initiation during the Stringent Response

      2016, Molecular Cell
      Citation Excerpt :

      The requirement for both DksA and RNAP for ppGpp crosslinking to DksA suggested that the second ppGpp binding site might be at the DksA-RNAP interface. Although there are no crystal structures of DksA-RNAP complexes, models based on genetic and biochemical studies suggest that DksA interacts with the β′ rim-helices at the entrance to the RNAP secondary channel, with its coiled-coil extending into the secondary channel and its coiled-coil tip in close proximity to the RNAP active site (Rutherford et al., 2009; Lennon et al., 2012; Lee et al., 2012; Furman et al., 2013; Parshin et al., 2015). To define RNAP residues in site 2, we initially screened RNAP variants that lacked site 1 and contained single substitutions for one of seven arginine or lysine residues in β′ near the DksA-RNAP interface in models of the complex (Figure 2A and legend).

    View all citing articles on Scopus
    View full text