The bridge helix coordinates movements of modules in RNA polymerase

Abstract

The RNA polymerase 'bridge helix' is a metastable α-helix that spans the leading edge of the enzyme active-site cleft. A new study published in BMC Biology reveals surprising tolerance to helix-disrupting changes in a region previously thought crucial for translocation, and suggests roles for two hinge-like segments of the bridge helix in coordinating modules that move during the nucleotide-addition cycle.

See Research article: http://www.biomedcentral.com/1741-7007/8/134

DNA-dependent, multisubunit RNA polymerases are conserved in core structure and are required for gene expression and regulation in all cellular organisms. To accomplish these roles, RNA polymerase has evolved into a complex molecular machine in which precisely orchestrated movements in a network of flexible modules mediate steps in a nucleotide-addition cycle of four basic steps: translocation of RNA and DNA chains through the polymerase; binding of the nucleotide triphosphate (NTP) substrate; catalysis; and pyrophosphate release. A key component of this network is the bridge helix, which occupies a critical position spanning the main channel of RNA polymerase just downstream of the active site (Figure 1a). After the first crystal structures of RNA polymerases emerged, the bridge helix garnered immediate attention as a possible effector of translocation, both because of its central location and because in RNA polymerases that are not bound to DNA, it partially unfolds to form a loop that clashes with the position of the templating DNA base in DNA-containing RNA polymerase structures (Figure 1b) [1]. Formation of the bridge helix loop is proposed to act as the pawl in a ratchet-like translocation mechanism to move DNA through RNA polymerase [2]. Subsequent crystal structures of NTP-bound elongation complexes suggested that the bridge helix might also play a role in catalysis [3–5]. In such structures, a continuous bridge helix forms a three-helix bundle with a neighboring domain known as the trigger loop, which folds into the trigger helices that contact NTP substrate in an NTP-bound elongation complex. Because positioning of the NTP substrate by trigger-helix contacts is required for efficient catalysis, even small movements of the bridge helix, not necessarily involving unfolding, may modulate catalysis by favoring or disfavoring formation of the trigger helices [3, 5].

Figure 1

Positions and conformations of the bridge helix in an elongation complex. (a) Structure of an elongation complex based on the crystal structure of a NTP-bound RNA polymerase from Thermus thermophilus (PDB 2o5j) [3]. DNA (black) is melting into a transcription bubble that allows template-strand pairing with RNA (red) in a 9-10 base pair RNA-DNA hybrid. The bridge helix (cyan) and trigger loop/helices (yellow/orange) lie on the downstream side of the active site. The presumed path of NTP entry is indicated by the straight arrow. Interconversion of the trigger loop and trigger helices is indicated by the curved arrow. The RNA polymerase subunits are shown as semi-transparent surfaces with the identities of orthologous subunits in bacteria (α, β, and β', gray, blue, and pink, respectively), archaea (D, L, B, and A), and eukaryotic RNA polymerase II (RPB3, 11, RPB2, RPB1) indicated. The active site Mg²⁺ ions are shown as yellow spheres, and α,β-methylene-ATP in green and red. (b) Conformations of the bridge helix observed on crystal structures of a NTP-bound elongation complex and of an RNA polymerase lacking nucleic acids. The positions of nascent RNA, the template DNA strand, α,β-methylene-ATP, Mg²⁺, and straight bridge helix are from the PDB 2o5j structure. The looped-out bridge helix indicating the conformation in a nucleic-acid-free structure is from T. thermophilus RNA polymerase bound by σ^A initiation factor (PDB 1iw7). Positions at which substitutions with proline increase polymerase activity are marked by Cα spheres (H_N and H_C) [7]. The location of a deletion of two amino acids in the plant RNA polymerase IV enzyme is marked by Cα-Cβ sticks (next to the white sphere marking the proline substitution). Sequences of the bridge helix from several RNA polymerases are shown, with the M. jannaschii bridge helix color-coded as in the molecular model: blue, segments in which two-amino-acid deletions eliminate polymerase activity; gray, segment in which deletions partially affect activity; white, segment in which deletions have minimal effect on activity; cyan, amino- and carboxy-terminal segments. Naturally occurring prolines at H_N and H_C are shown white-on-black.

Testing the contributions to RNA polymerase function of these two proposed actions of the bridge helix (which are not mutually exclusive) - or revealing other bridge-helix roles - is made difficult by the small movements involved relative to the size of the polymerase and by the inability of crystal structures to report molecular dynamics. Over the past few years, Weinzierl and colleagues have developed and exploited a novel approach that augments conventional structure-function studies by assaying RNA polymerase with systematically altered bridge-helix structures [6]. This ambitious undertaking was accomplished using robotics to assemble and assay many variants of an archaeal RNA polymerase (from Methanocaldococcus jannaschii) that can be reconstituted in vitro from individual subunits. The most recent results from this systematic dissection of the bridge helix, published in BMC Biology by Weinzierl [7], suggest that kinking of the helix at or adjacent to segments that contact the interconnected network of RNA polymerase modules may play a more important role in the nucleotide-addition cycle than contacting the template base or looping to generate a translocation pawl.