Regulation through the RNA Polymerase Secondary Channel: STRUCTURAL AND FUNCTIONAL VARIABILITY OF THE COILED-COIL TRANSCRIPTION FACTORS

Jindrich Symersky,Anna Perederina,Marina N Vassylyeva,Vladimir Svetlov,Irina Artsimovitch,Dmitry G Vassylyev

doi:10.1074/jbc.c500405200

Abstract

Gre factors enhance the intrinsic endonucleolytic activity of RNA polymerase to rescue arrested transcription complexes and are thought to confer the high fidelity and processivity of RNA synthesis. The Gre factors insert the extended alpha-helical coiled-coil domains into the RNA polymerase secondary channel to position two invariant acidic residues at the coiled-coil tip near the active site to stabilize the catalytic metal ion. Gfh1, a GreA homolog from Thermus thermophilus, inhibits rather than activates RNA cleavage. Here we report the structure of the T. thermophilus Gfh1 at 2.4 A resolution revealing a two-domain architecture closely resembling that of GreA. However, the interdomain orientation is strikingly distinct (approximately 162 degrees rotation) between the two proteins. In contrast to GreA, which has two acidic residues on a well fixed self-stabilized alpha-turn, the tip of the Gfh1 coiled-coil is flexible and contains four acidic residues. This difference is likely the key to the Gre functional diversity, while Gfh1 inhibits exo- and endonucleolytic cleavage, RNA synthesis, and pyrophosphorolysis, GreA enhances only the endonucleolytic cleavage. We propose that Gfh1 acidic residues stabilize the RNA polymerase active center in a catalytically inactive configuration through Mg2+-mediated interactions. The excess of the acidic residues and inherent flexibility of the coiled-coil tip might allow Gfh1 to adjust its activity to structurally distinct substrates, thereby inhibiting diverse catalytic reactions of RNA polymerase.

Highlights

IntroductionThe bacterial factors GreA, GreB, and DksA all employ a long ␣-helical coiled-coil (CC) domain protruding through the secondary channel to target functionally crucial Mg2ϩ ions near the RNA polymerase (RNAP) active site (AS) with the two invariant acidic residues at the CC tips (8 –10, 12)
A low resolution (15 Å) electron density [12] demonstrates that the RNA polymerase (RNAP) secondary channel accommodates the GreB CC-domain but does not allow one to distinguish between the alternative G-domain orientations: the GreA and Gfh1 G-domains would likely fit well to the same electron density if the proteins are superimposed by their CC-domains
The published models for RNAP-Gre factor interactions agree on the overall topology of the complex but differ in their details [8, 10, 12], and substitutions in RNAP that directly affect the Gre factors binding have not been reported

Summary

Introduction

The bacterial factors GreA, GreB, and DksA all employ a long ␣-helical coiled-coil (CC) domain protruding through the secondary channel to target functionally crucial Mg2ϩ ions near the RNAP AS with the two invariant acidic residues at the CC tips (8 –10, 12) Despite this structural similarity, the functions of these proteins are strikingly distinct; the Gre factors directly remodel the RNAP AS to stimulate the endonucleolytic cleavage of the RNA [8, 10, 12], whereas DksA amplifies the activity of the “magic spot” (ppGpp), the regulator of stringent response in bacteria, but has no direct effect on catalysis [9, 13]. Despite a significant sequence conservation including the two mechanistically important acidic residues (Fig. 1A), this Gre-like factor does not stimulate but instead inhibits the intrinsic and GreA-mediated endonucleolytic RNA cleavage [14] as well as the RNA synthesis [15]

Methods

Results

Conclusion