Journal of Molecular Biology
An Insertion in the Catalytic Trigger Loop Gates the Secondary Channel of RNA Polymerase
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.).
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2018, Molecular CellCitation 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
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