Structure-based analysis of RNA polymerase function: the largest subunit's rudder contributes critically to elongation complex stability and is not involved in the maintenance of RNA-DNA hybrid length

Abstract

contributed equally to this work Analysis of multisubunit RNA polymerase (RNAP) structures revealed several elements that may constitute the enzyme's functional sites. One such element, the`rudder', is formed by an evolutionarily conserved segment of the largest subunit of RNAP and contacts the nascent RNA at the upstream edge of the RNA±DNA hybrid, where the DNA template strand separates from the RNA transcript and re-anneals with the non-template strand. Thus, the rudder could (i) maintain the correct length of the RNA±DNA hybrid; (ii) stabilize the nascent RNA in the complex; and (iii) promote or maintain localized DNA melting at the upstream edge of the bubble. We generated a recombinant RNAP mutant that lacked the rudder and studied its properties in vitro. Our results demonstrate that the rudder is not required for establishment of the upstream boundary of the transcription bubble during promoter complex formation, nor is it required for separation of the nascent RNA from the DNA template strand or transcription termination. Our results suggest that the rudder makes critical contributions to elongation complex stability through direct interactions with the nascent RNA.

The interaction between RNA polymerase and the elongation factor NusA

“…5,23,24 This would allow NusA to bind resulting in a transition complex comprising both factors, 23 consistent with data derived from in vitro experiments. 19 Binding of NusA is likely to induce structural changes in RNAP, which in turn could further reduce the affinity of σ interaction to RNAP.…”

“…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. 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.…”

Altering the interaction between σ 70 and RNA polymerase generates complexes with distinct transcription-elongation properties

“…However, the effect of this interaction on RNA polymerase has not been determined. The coiled-coil supports the rudder, which interacts with nascent RNA at the upstream edge of the DNA͞RNA hybrid (54), an interaction believed to stabilize the elongation complex (55). The altered interaction between the coiled-coil and region 2.2 of 70(E407K) could change either the initial positioning or conformation of the rudder.…”

Relations Between Replication and Transcription

“…In simple terms, if using a comparison of RNAP to a crab claw, the claw in initiation is more open, while in elongation it closes on the RNA-DNA hybrid almost fully surrounding it. These interactions of RNAP with nucleic acids make elongation complex highly stable and resistant to very high ionic strength (1 M KCl) and to competitors (Kuznedelov et al 2002). The structure of the elongation complex is schematically shown in Figure 1.…”