Structures of the RNA polymerase-σ54 reveal new and conserved regulatory strategies

See allHide authors and affiliations

Science  21 Aug 2015:
Vol. 349, Issue 6250, pp. 882-885
DOI: 10.1126/science.aab1478

Keeping gene transcription in check

Transcription of all genes is carried out by RNA polymerase (RNAP). The enzyme is thus a pivotal regulation point for many cell and developmental processes. In bacteria, sigma factors play a vital role in transcription regulation, with σ54 being critical for transcription of many stress response genes. Yang et al. determined the x-ray crystal structure of RNAP bound to σ54, as well as promoter DNA. In the initial inhibited state of the RNAP-σ54 complex, the σ54 blocks the template DNA from entering the RNAP active site and the downstream DNA channel.

Science, this issue p. 882


Transcription by RNA polymerase (RNAP) in bacteria requires specific promoter recognition by σ factors. The major variant σ factor (σ54) initially forms a transcriptionally silent complex requiring specialized adenosine triphosphate–dependent activators for initiation. Our crystal structure of the 450-kilodalton RNAP-σ54 holoenzyme at 3.8 angstroms reveals molecular details of σ54 and its interactions with RNAP. The structure explains how σ54 targets different regions in RNAP to exert its inhibitory function. Although σ54 and the major σ factor, σ70, have similar functional domains and contact similar regions of RNAP, unanticipated differences are observed in their domain arrangement and interactions with RNAP, explaining their distinct properties. Furthermore, we observe evolutionarily conserved regulatory hotspots in RNAPs that can be targeted by a diverse range of mechanisms to fine tune transcription.

Gene transcription is a tightly regulated event. A range of factors are required to maintain RNAP in a signal-responsive, but inhibited, state and to target it to specific genes (1). Bacterial sigma (σ) factors, eukaryotic TFIIB, and other general transcription factors, are the primary promoter recruitment factors. The major variant σ54 (also called σN) is used in transcribing genes for numerous stress responses (2). Unlike the major σ70 class, which recognizes the –35 and –10 promoter DNA elements, the σ54 class directs RNAP to promoter sites through the –24 and –12 DNA elements and forms a stable closed promoter complex that is unable to spontaneously melt DNA and initiate transcripts (3). Instead, the initiation process requires adenosine triphosphate (ATP)–dependent activator proteins bound to upstream enhancer sites to actively remodel the RNAP-σ54–DNA complex (4).

Our crystal structure of RNAP-σ54 reveals that σ54 contains four structural domains connected by long coils and loops that span a large area of the RNAP core enzyme, which consists of two α and β, β′, and ω subunits (5, 6) (Fig. 1, A to C; fig. S1, A and B; and table S1). σ54 Region I (RI, residues 1–56) forms a hook composed of two α helices. Region II (RII, residues 57–120) also contains two α helices (residues 57–85) in addition to loops that are buried inside the RNAP (Fig. 1, A and C). The RNAP core-binding domain (CBD) of σ54 extends as a structural fold to residue 250 and consists of two α-helical subdomains. Following on from the CBD, the backbone extends back to connect to a loop region; before an extra-long α helix (residues 315–353, hereafter called ELH, spanning 50 Å); followed by the HTH domain (residues 365–385), involved in interaction with the –12 promoter elements (3). The domain containing the RpoN box (RpoN domain), which is responsible for recognizing the –24 promoter elements, consists of a three-helical bundle (residues 415–477) (Fig. 1A) (3). The σ54 polypeptide chain snakes back and forth through its loop regions embedded in the RNAP. We carried out cross-linking experiments between σ54 and RNAP in solution using a p-benzoyl-l-phenylalanine–incorporated RNAP library. The amino acids that are cross-linked to σ54 agree well with the RNAP-σ54 crystal structure (Fig. 1D, yellow, and fig. S1C).

Fig. 1 Structure of complete RNAP-σ54.

(A) Structure and schematic diagram of σ54. (B) Structure of σ70 (PDB 4YG2) with similar functional domains colored accordingly. (C) Holoenzyme in different orientations, RNAP core in cylinders. β, cyan; β′, salmon pink; α, gray; ω, pale blue; σ54 in ribbon and colored as in (A); DS, downstream; US, upstream. (D) Residues of β and β′ subunits that are cross-linked to σ54 are mapped onto the structure and colored in yellow. (E) Electron density map and structural model of RNAP-σ54–DNA.

We crystallized RNAP-σ54 in complex with a 28–base pair (bp) DNA containing the –24 and –12 promoter elements. The crystals diffracted to 8 Å resolution, the electron density for DNA was unambiguous (Fig. 1E), and the molecular envelope for the holoenzyme was clear. We thus constructed a structural model of RNAP-σ54–DNA (Fig. 1E and fig. S1D).

All σ factors contain a major RNAP CBD and a major DNA binding domain, which recognize either the –35 or –24 elements. The CBD of σ54 (Fig. 1A) binds toward the upstream face of the RNAP (Fig. 1C) (on the basis of the promoter DNA binding orientation), which interacts extensively with many functional modules within the RNAP, including the β flap (residues 835–937), the C terminus of the β subunit (residues 1267–1320), the β′ zipper/Zn-binding domain (residues 35–100), and the β′ dock domain (residues 370–420), as well as the α-subunit carboxyl-terminal domain (α-CTD) (Fig. 2A). Using FeBABE cleavage assays, we have previously mapped residue 198 of σ54 (within the CBD) to a region within the β flap (7).

Fig. 2 Functional domains of σ54 and interactions with RNAP core.

(A) Detailed view of the CBD and RNAP interactions. (B) RI and RIII. (C) DNA-RNA channels, with DNA, orange, and RNA, magenta. (D) Overlay with RII.1, RII.2, and RII.3.

The RpoN domain of σ54 is the most conserved domain among σ54 from different organisms (fig. S2). This domain extends from the main body of RNAP and does not contact other parts of σ54 or core enzyme (Fig. 1, A and C). Instead, it interacts with an adjacent RNAP molecule in the crystal, which suggests that its location is flexible in solution. In the RNAP-σ54–DNA complex model, the RpoN domain is indeed moved relative to the RNAP-σ54 structure (fig. S1D). Unlike the flexible RpoN domain, the RIII-HTH domain of σ54, which binds to the –12 promoter region, stably associates with the RNAP core through RI–RIII (Fig. 1E and fig. S1D) (810). Our data thus suggest that –12 binding is a major functional determinant for the σ54 holoenzyme promoter recognition, as well as a stable closed complex formation.

The σ54 RI plays an inhibitory role and contains contact sites for its cognate activator proteins (11, 12). RI interacts with RIII, forming a structural module that lies along the cleft between β and β′ (Figs. 1C and 2B), where template strand DNA enters the active-site cleft (13). Our structure clearly suggests that the entry of promoter DNA into RNAP is blocked by RI and RIII of σ54. The way the RNAP-σ54 crystal structure fits into the cryoelectron microscopy map of activator-bound RNAP-σ54 holoenzyme (14) positions the RI helices inside the connecting density leading to the activator protein, which indicates that RI in the RNAP-σ54 structure is presented favorably to contact the activator (fig. S3A).

RII penetrates deeply into the DNA binding channel (Fig. 1 and Fig. 2, C and D). RII can be divided into three subregions based on their locations in the holoenzyme. RII.1 (residues 57–85) occupies the downstream DNA binding cleft of RNAP, just above the bridge helix, and thus plays an inhibitory role (Fig. 1 and Fig. 2, C and D, and fig. S3A, right). RII.2 (residues 86–105) occupies the space of the DNA template strand (Fig. 2, C and D), which indicates that RII.2 has to relocate to permit template-strand DNA access into the RNAP active site and for transcription initiation. RII.3 (residues 105–120) extends along the path that is occupied by RNA in the transcribing complex (Fig. 2, C and D), which suggests that it will also need to relocate upon RNA synthesis.

Comparisons between σ54 and σ70 holoenzyme structures reveal that, overall, they contact similar regions of the RNAP core enzyme (Fig. 3, A and B), in agreement with the idea that specific factors unrelated by structure and sequence can be functionally similar (15). However, the relative location of functional domains is completely different. For example, the σ54 CBD is located upstream, which blocks the RNA exit tunnel (Fig. 3C), whereas σ2, the major CBD of σ70, contacts the downstream β′ region (Fig. 3, A and B). The structural differences suggest that σ54 CBD, and hence σ54, will have to dissociate or relocate to allow progression from transcription initiation to elongation, whereas σ70 can loosely associate with the RNAP core even during transcript elongation, in agreement with earlier biochemical data (16, 17). σ70 region 4 (σ4), which recognizes the –35 promoter element, occupies the upstream surface blocking the RNA exit channel instead (Fig. 3B).

Fig. 3 Comparisons of σ54 and σ70 holoenzymes.

(A) RNAP-σ54 with RNAP displayed as spheres and σ54 as surface. (B) RNAP-σ70, same view as in (A). (C) Elongation complex (PDB code 2O5J).

The inhibitory RI of σ54 interacts with RIII across the RNAP cleft and blocks the template strand from entering. On the other hand, σ1.1 of σ70, which also has an inhibitory role (18), is located in the downstream DNA channel, overlapping with the region occupied by σ54 RII.1 (Fig. 3), which suggests that σ1.1 and RII.1 might play related roles in transcription regulation. However, σ1.1 connects with σ2 by only a single flexible linker, which allows the σ1.1 to move readily from the RNAP cleft (Fig. 1B, Fig. 3B). σ54 RII.1 is connected to the RI and CBD. RI interacts with RIII-ELH, whereas the linker that connects RII.1 with the CBD is embedded in the DNA/RNA channel of RNAP (Fig. 2, C and D). Because of its topological restraint, σ54 RII.1 cannot relocate easily without affecting the conformation of other parts of σ54; RII.1 will thus block downstream DNA entry.

Notable conformational differences exist between the σ70 and σ54 holoenzymes in many key modules within the RNAP when superposed on the bridge helix, chosen because the catalytic center must be conserved (fig. S3B). The β′ coiled coil, with the whole β′ subunit, is rotated, which narrows the downstream DNA channel by as much as 5 Å (fig. S3B). However, the clamp, which consists of β and β′ domains forming the walls of the downstream DNA channel, was shown to adopt multiple conformations in solution (19). The β′ coiled coil forms helical bundles with σ2 of σ70 and helps to stabilize the unpaired nontemplate DNA strand and, hence, the transcription bubble (20) (fig. S4A). The B linker of TFIIB also interacts with the β′ coiled coil (fig. S4B). In the σ54 holoenzyme structure, the β′ coiled coil does not form a helical bundle with σ54 (fig. S4C). However, it is possible that enroute to transcription initiation, this interaction is established, in agreement with our biochemical data (fig. S4D).

σ54 occupies multiple locations along the DNA and RNA path within the RNAP core. These locations are also targeted by other regulatory factors and in other RNAPs. For example, RI and RIII sit across the RNAP cleft, blocking the DNA template strand entry (Fig. 4A). At similar locations, domains 2 and 3 (σ2 and σ3) of σ70 form a V-shaped wedge that acts as a gate to allow the template strand to enter the active cleft (Fig. 4B) (13, 21). TFIIB also uses two subdomains, the B core and the B linker, to occupy similar areas in Pol II (Fig. 4C). This area is thus occupied by σ54 to inhibit, while guiding the template strand delivery by σ70 and TFIIB. Furthermore, σ2 of σ70 contains a number of aromatic residues that are shown to interact with the nontemplate strand and therefore facilitate DNA melting and transcription bubble formation (20). In σ54, there are few aromatic residues in this region (RI and RIII–ELH) that could facilitate DNA melting, in agreement with data showing that although deletion of σ54 RI can bypass the requirement of activator proteins, it fails on double-stranded DNA (11). Activator proteins may therefore be directly required for transcription bubble opening. The downstream DNA channel is occupied by both RII.1 of σ54 and σ1.1 of σ70 (Fig. 4D). A negatively charged region of TFIIF is also located in the downstream DNA channel of Pol II (22). RII.2 of σ54 is located in the channel that is occupied by the template strand DNA in the transcribing RNAP (Fig. 2D). Both σ70 and TFIIB lack equivalent structural features. This area, however, is occupied in Pol I by part of the expander (Fig. 4E), a unique insertion in Pol I compared with Pol II and shown to stabilize an expanded active center cleft (23, 24) (Fig. 4E). Similar to RII.3, both region 3.2 in σ70 (the σ finger) and the B reader in TFIIB are shown to overlap with the space that would be occupied by RNA (Fig. 4, D and F) (25). Indeed, RII.3 shares sequence homology with the σ finger, especially the highly conserved DDE motif (fig. S2). Acidic residues in these elements are proposed to facilitate template DNA loading and RNA separation from DNA and to guide it toward the exit channel (2528). It is possible that RII.3 also performs similar roles.

Fig. 4 Regulatory elements of σ54 and comparison with σ70, TFIIB, and Pol I.

(A) σ54, (B) σ70, and (C) TFIIB in relation to template strand (gold), as defined in the complex with a full transcription bubble (4YLN). (D to F) Comparison of σ54 with σ70, Pol I (4C2M), and TFIIB (4BBS) in the RNAP DNA-RNA channel; structures are superposed on the bridge helix. σ54 represented as spheres; σ70, TFIIB, and Pol I expander are shown as ribbons; and RNAP as cylinders.

Our structure uncovers a diverse range of regulatory strategies used by σ54 in tightly controlling transcription initiation. These include blocking the template strand from entering the RNAP active cleft by RI and RIII, occupying the downstream DNA channel by RII.1, and interfering with the template strand and synthesized RNA by RII.2–RII.3. The position of RI and RIII plays a vital role in these regulatory functions. Activator proteins interact with RI and could relocate RI, RIII–ELH, and RII and release the inhibition posed by some of these structural elements (fig. S3A) (29, 30). Precisely how activators overcome these multiple modes of inhibition, as well as the role of ATP in this process, remains to be determined. Furthermore, we show that although σ54, σ70, TFIIB, TFIIF, and Pol I subunits have no sequence or structural similarities, they target the same elements within their respective RNAPs. Our structure and comparisons thus provide clear evidence that regulatory hotspots within RNAPs are functionally conserved. Different transcription factors, irrespective of their structures and sequences, are used to target these hotspots in order to exert a fine-tuned transcription regulation strategy (fig. S5). It will be interesting to see the extent to which additional multisubunit RNAPs use these regulatory strategies.

Supplementary Materials

Materials and Methods

Figs. S1 to S5

Tables S1

References (3143)


  1. Acknowledgments: We thank M. Michael and D. Bose for their earlier contributions to this project; A. Forster, J. Liu, and beamline scientists at the Diamond Light Source for their help with data collection; and members of X.Z.’s and M.B.’s labs for helpful discussion. We thank D. Wigley, R. Dixon, R. Weinzierl, C. Fernández-Tornero, and R. Wigneshweraraj for critically reading the manuscript. Y.Y. was funded by the Chinese National Science Foundation and the China Scholarship Council. The majority of this work was funded by the UK Biotechnology and Biological Sciences Research Council to X.Z. and M.B. K.S.M. is supported by NIH grant GM087350. Y.-P.W. is funded by 973 National Key Basic Research Programme (2015CB755700) in China. J.T.W. and R.L.G. were supported by NIH grant R37 GM37048 (to R.L.G.). The atomic coordinate has been deposited in the Protein Data Bank with accession code 5BYH.

Stay Connected to Science

Navigate This Article