RNA polymerase gate loop guides the nontemplate DNA strand in transcription complexes

Abstract

Upon RNA polymerase (RNAP) binding to a promoter, the σ factor initiates DNA strand separation and captures the melted nontemplate DNA, whereas the core enzyme establishes interactions with the duplex DNA in front of the active site that stabilize initiation complexes and persist throughout elongation. Among many core RNAP elements that participate in these interactions, the β′ clamp domain plays the most prominent role. In this work, we investigate the role of the β gate loop, a conserved and essential structural element that lies across the DNA channel from the clamp, in transcription regulation. The gate loop was proposed to control DNA loading during initiation and to interact with NusG-like proteins to lock RNAP in a closed, processive state during elongation. We show that the removal of the gate loop has large effects on promoter complexes, trapping an unstable intermediate in which the RNAP contacts with the nontemplate strand discriminator region and the downstream duplex DNA are not yet fully established. We find that although RNAP lacking the gate loop displays moderate defects in pausing, transcript cleavage, and termination, it is fully responsive to the transcription elongation factor NusG. Together with the structural data, our results support a model in which the gate loop, acting in concert with initiation or elongation factors, guides the nontemplate DNA in transcription complexes, thereby modulating their regulatory properties.D uring each round of transcription, RNA polymerase (RNAP) establishes, maintains, and finally releases contacts with the DNA template and the RNA. These interactions mediate highly selective initiation, processive elongation, and precise termination and can be tuned to enable intricate regulation during the transcription cycle. RNAP resembles a crab claw in which the two pincers composed of the β′ and β subunits form an active site cleft that accommodates the nucleic acid chains (Fig. 1). The mobile β′ clamp domain that forms one of the pincers stands out as a central regulatory feature (1,2). The open clamp likely allows the loading of the promoter DNA during initiation and the release of the template and the nascent RNA during termination. The clamp is thought to close to form an active initiation complex and to remain closed during elongation but may open partially at a hairpindependent pause site (3).The clamp movements may be linked to those of the β lobe domain, which forms a part of the second pincer. The β gate loop (GL), which lies across the RNAP cleft from the tip of the clamp (Fig. 1), has been identified as an element that restricts the entry of the duplex promoter DNA into the narrow active site cleft (4), allowing only a single strand of DNA to pass through. This model posited that the DNA stands must separate outside the cleft before entry into RNAP, whereas footprinting studies demonstrated that the promoter DNA enters the active site cleft before it is opened (5). This controversy was a subject of an intense debate until a recent study revealed that the...

Locking the nontemplate DNA to control transcription

“…NusG/Spt5 proteins bind to the elongating RNAP and favor productive RNA synthesis. While most discussion has focused on similar interactions of the NTD with RNAP, widely assumed to be responsible for the antipausing activity of all members of this family, the role of direct DNA contacts has only recently come to light (Crickard et al ., ; NandyMazumdar et al ., ; Yakhnin et al ., ). Our present results argue that remodeling of the NT DNA and the upstream DNA duplex by RfaH makes a significant contribution to its effects on RNA chain elongation.…”

“…However, recent findings put this model in doubt and suggest that NusG‐like proteins may utilize other contacts with the EC to reduce pausing. First, the bridging contacts to GL do not appear to be universally important; E. coli NusG does not require GL for antipausing (NandyMazumdar et al ., ) and antibacktracking (Turtola and Belogurov, ) activities. Second, in cryo‐EM structures of the hairpin‐stabilized paused E. coli ECs (Guo et al ., ; Kang et al ., a), the clamp does not open, and instead undergoes a minor rotation termed ‘swiveling’.…”

“…E. coli RfaH (Artsimovitch and Landick, ), Bacillus subtilis NusG (Yakhnin et al ., ) and Saccharomyces cerevisiae Spt5 (Crickard et al ., ) have been shown to directly contact NT DNA. We proposed that NusG‐DNA contacts compensate for missing contacts with GL (NandyMazumdar et al ., ), providing alternative means to stabilize the NT DNA‐GL‐NTD network of interactions. We hypothesized that excessive flexibility of NT DNA could be detrimental, and that a factor that restrains NT DNA may promote processive elongation (NandyMazumdar et al ., ); a similar ‘bubble‐chaperone’ model was proposed for yeast Spt5 (Crickard et al ., ).…”

“…We proposed that NusG‐DNA contacts compensate for missing contacts with GL (NandyMazumdar et al ., ), providing alternative means to stabilize the NT DNA‐GL‐NTD network of interactions. We hypothesized that excessive flexibility of NT DNA could be detrimental, and that a factor that restrains NT DNA may promote processive elongation (NandyMazumdar et al ., ); a similar ‘bubble‐chaperone’ model was proposed for yeast Spt5 (Crickard et al ., ). Finally, interactions of NusG proteins with the upstream DNA duplex and the fork junction may facilitate forward translocation to reduce pausing and arrest.…”

Reading of the non‐template DNA by transcription elongation factors

“…These data are a strong indicator that NT toggling between flexible and restrained states is the physical switch between the pausing‐prone and pausing‐resistant states of TEC. Dependence of this transition on RfaH‐RNAP [mainly the RNAP gate loop (NandyMazumdar et al ., )] interactions was also demonstrated, providing further support of a model in which the NTD interactions with both DNA and RNAP are required for RfaH recruitment to the TEC and the subsequent rendering of the TEC resistant to pausing and termination.…”