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

Abstract

Bacterial Gre transcript cleavage factors stimulate the intrinsic endonucleolytic activity of RNA polymerase (RNAP) to rescue stalled transcription complexes. They bind to RNAP and extend their coiled-coil (CC) domains to the catalytic centre through the secondary channel. Three existing models for the Gre-RNAP complex postulate congruent mechanisms of Gre-assisted catalysis, while offering conflicting views of the Gre-RNAP interactions. Here, we report the GreB structure of Escherichia coli. The GreB monomers form a triangle with the tip of the amino-terminal CC of one molecule trapped within the hydrophobic cavity of the carboxy-terminal domain of a second molecule. This arrangement suggests an analogous model for recruitment to RNAP. Indeed, the beta'-subunit CC located at the rim of the secondary channel has conserved hydrophobic residues at its tip. We show that substitutions of these residues and those in the GreB C-terminal domain cavity confer defects in GreB activity and binding to RNAP, and present a plausible model for the RNAP-GreB complex.

Figure 1

Structure of the GreB protein. (A) Overall structure. The α-helical turn at the tip of the N-domain and the two principal acidic side chains are shown in green. (B) Structures of GreB and GreA superimposed by the C-domains. (C) Sequence alignment of the Gre factors. The principal acidic residues and the conserved hydrophobic residues lining the C-domain cavity are highlighted in green and magenta, respectively. We included serine and threonine, whose small polar side chains often appear in the hydrophobic cores of proteins, as residues that contribute to the hydrophobic intermolecular interface, as observed in the GreB trimers (D). GreB residues that were altered in the present study are marked by red asterisks. (D) Hydrophobic interface formed between two GreB molecules in the asymmetric unit. (E) Overall view of the trimer that the GreB monomers form in the crystal. C-domain, carboxy-terminal domain; N-domain, amino-terminal domain.

Figure 2

Functional analysis of the putative β′-subunit coiled coil–GreB interface. (A) Sequence alignment of the tip of the β′CC. The conserved residues that might constitute the hydrophobic intermolecular interface with GreB are highlighted in orange. The double-substituted tip residues are indicated by red asterisks. (B) Effects of the β′CC substitutions on RNA cleavage in scaffold complexes (used because the deletion enzyme was incapable of initiation at the λP_R promoter). Left panel: a 15% representative gel with the 3′-end-labelled 14-mer RNA and a 2-mer 3′-cleavage product indicated. TECs assembled with core enzymes were incubated at 37°C for the durations indicated above (in min) in the absence or presence of 100 nM of wild-type GreB. Right panel: quantification of the 14-mer RNA that remained uncleaved after 5 min incubation. The assay was repeated at least twice for each enzyme variant. (C) Gel shift assay with radiolabelled GreBs (at 8 nM) and increasing concentrations of core RNAP (0, 40, 80, 150, 300, 500 and 1,000 nM). Positions of the wells and the Gre–RNAP complexes are indicated. The core concentration at which approximately 50% of ³²P-GreB was bound is shown; no binding was detected even at 1 μM of the DD core (as well as the RR and deletion variants; data not shown). (D) Effects of the selected GreB variants on RNA cleavage in halted radiolabelled A26 TECs. The fraction of A26 RNA remaining after 10 min incubation at 37°C is shown below each lane. The far left lane contains A26 RNA before 37°C incubation. AA, double alanine substitution; β′CC, β′-subunit coiled coil; DD, double aspartic acid substitution; RNAP, RNA polymerase; RR, double arginine substitution; TEC, transcription elongation complex; WT, wild-type.

Figure 3

Structural model of the RNAP–GreB complex. (A) Overall view of the complex. The structure of the Thermus thermophilus RNAP (Vassylyev et al, 2002), in which the β′CC tip residues were substituted for the Escherichia coli counterparts, was used for the modelling. (B) Three-dimensional view of the hypothetical hydrophobic interface and (C) a schematic drawing of possible van der Waals contacts (dashed lines) between the GreB (magenta) and β′CC (yellow) residues. (D) The principal acidic residues (green) at the GreB N-domain tip are positioned at the interacting distance with the active site. The blue dashed lines indicate the shortest distances between the GreB Asp 41 (3.7 Å) and Glu 44 (3.4 Å) and the major (high affinity) catalytic Mg²⁺ ion (cMG1), suggesting that these side chains would be able to coordinate the second catalytic Mg²⁺ ion, the precise position of which in the active site of RNAP has not yet been determined. β′CC, β′-subunit coiled coil; C-domain, carboxy-terminal domain; cMG1, a high-affinity catalytic Mg²⁺ ion in the RNAP active site; N-domain, amino-terminal domain; RNAP, RNA polymerase.