The structure of bacterial RNA polymerase in complex with the essential transcription elongation factor NusA

Abstract

There are three stages of transcribing DNA into RNA. These stages are initiation, elongation and termination, and they are wellunderstood biochemically. However, despite the plethora of structural information made available on RNA polymerase in the last decade, little is available for RNA polymerase in complex with transcription elongation factors. To understand the mechanisms of transcriptional regulation, we describe the first structure, to our knowledge, for a bacterial RNA polymerase in complex with an essential transcription elongation factor. The resulting structure formed between the RNA polymerase and NusA from Bacillus subtilis provides important insights into the transition from an initiation complex to an elongation complex, and how NusA is able to modulate transcription elongation and termination.

The interaction between RNA polymerase and the elongation factor NusA

“…The resultant model ( Fig. 1) is consistent with the biochemical binding data, 22 and that of other previous biochemical observations (see discussion below), enabling refined hypotheses on how NusA plays its various regulatory roles in the transcription cycle.…”

“…22 To our surprise, NusA was found to bind to a completely different region on RNAP to the major σ interaction site (the CH region of the β' subunit). 5,23 Instead, NusA was shown to bind to the β-flap.…”

“…22 This conformation of NusA is highly bent in comparison to the crystal structures of NusA, but would be permitted by the inherent flexibility in the ∼23 amino acid linker between the N-and C-terminal domains. 12,13,18,22 This placement of NusA indicated that any mutually exclusive model for NusA-σ binding could not be accounted for by the two proteins competing for the σ region 2 binding site on the CH region of the β' subunit. However, since region 4 of σ also makes weak contacts with the β-flap tip of RNAP, 5 it could compete with NusA for binding to that region of RNAP.…”

“…This interaction occurs specifically between the N-terminal domain of NusA and the β-flap tip helix, while the C-terminal domain contributes to the stability of the complex through non-specific interactions with RNAP. 22 This unexpected result led us to undertake structural ensuring efficient termination at intrinsic terminators. 9 The bi-functional role of NusA has been shown to result from a change in the stoichiometry of NusA to RNAP from 1:1 during mRNA transcription, to 2:1 in the antitermination complexes.…”

“…8 This activity is important in poiNt of view poiNt-of-view studies to produce a model for RNAP in complex with NusA, where EM structures were resolved for both negatively stained RNAP core enzyme and a NusA-RNAP complex. 22 This represented the first structure of RNAP in complex with an essential transcription elongation factor. The resultant model ( Fig.…”