Structures of TECs bound to NusG and RfaH. The NTD inhibits RNAP backtracking, which can lead to arrest ( 22), while the CTD binds to ribosomal protein S10 to couple transcription to translation ( 23) or to Rho to terminate synthesis of antisense and foreign RNAs ( 24). NusG NTD exhibits a mixed α/β fold connected via a flexible linker to a β-barrel CTD ( 10, 20, 21). NusG is a housekeeping factor that binds to RNAP transcribing most genes ( 19) and is essential for cellular viability. Here, we investigate interactions of two best-studied members of this family, Escherichia coli NusG and RfaH, with RNAP (Figure (Figure1). Several RNAP inhibitors modulate clamp opening by binding to dynamic switch regions located at the base of the clamp domain ( 17, 18).
Examples of allosteric modulators of RNA synthesis include nascent RNA hairpins ( 11), termination factor Rho ( 12, 13), and antibiotics ( 14–16). In addition to this local bridging effect, the NTD binding to RNAP could trigger an allosteric signal affecting distant elements in the enzyme, such as the catalytic center located tens of Angstroms away. By bridging the gap between the RNAP pincers, NusG homologs would guard against accidental opening of the clamp and premature termination. The RNAP pincers open to load the duplex DNA during initiation and close around the DNA upon the transition to elongation prior to processive RNA synthesis. The ubiquitous ‘anti-pausing’ activity of isolated NTDs ( 4, 9, 10) is commonly explained by their ability to act like processivity clamps ( 8). Second, their C-terminal domains (CTDs, one in prokaryotes or several in eukaryotes) bind to diverse cellular partners to coordinate transcription with other processes, such as translation in prokaryotes or splicing in eukaryotes ( 1). First, their N-terminal domains (NTDs) bind to two mobile pincers of the elongating RNAP, the clamp and the lobe/protrusion domains ( 2–7), completing the circle around the nucleic acids to promote productive RNA synthesis ( 8).
NusG-like proteins have two key functions.
Our results provide insights into differences in structural dynamics exerted by NusG and RfaH during binding to TEC, which may explain their different functional outcomes and allosteric regulation of transcriptional pausing by RfaH.Ĭellular RNA polymerases (RNAP) are multi-domain enzymes that transcribe the genomes in every domain of life, and their activities are elaborately controlled by a plethora of accessory proteins, among which NusG family is the only universally conserved group of transcription factors ( 1). Additional changes far from the factor-binding site were observed only with RfaH. We found that NusG and RfaH regions that bind RNAP became solvent-protected in factor-bound TECs, whereas RNAP regions that interact with both factors showed opposite deuterium uptake changes when bound to NusG or RfaH. Here, we employed hydrogen-deuterium exchange mass spectrometry to investigate changes in local and non-local structural dynamics of Escherichia coli NusG and its paralog RfaH, which have opposite effects on expression of xenogenes, upon binding to TEC.
Some of these differences could be due to conformational changes in RNAP and NusG-like proteins, which cannot be captured in static structures. However, NusG homologs differ in their regulatory roles, modes of recruitment, and effects on RNA synthesis. Structures of factor-bound transcription elongation complexes (TECs) reveal similar contacts to RNAP, consistent with a shared mechanism of action. In every domain of life, NusG-like proteins bind to the elongating RNA polymerase (RNAP) to support processive RNA synthesis and to couple transcription to ongoing cellular processes.
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