Regulation of transcriptional pausing through the secondary channel of RNA polymerase

Abstract

Transcriptional pausing has emerged as an essential mechanism of genetic regulation in both bacteria and eukaryotes, where it serves to coordinate transcription with other cellular processes and to activate or halt gene expression rapidly in response to external stimuli. Deinococcus radiodurans, a highly radioresistant and stressresistant bacterium, encodes three members of the Gre family of transcription factors: GreA and two Gre factor homologs, Gfh1 and Gfh2. Whereas GreA is a universal bacterial factor that stimulates RNA cleavage by RNA polymerase (RNAP), the functions of lineage-specific Gfh proteins remain unknown. Here, we demonstrate that these proteins, which bind within the RNAP secondary channel, strongly enhance site-specific transcriptional pausing and intrinsic termination. Uniquely, the pause-stimulatory activity of Gfh proteins depends on the nature of divalent ions (Mg 2+ or Mn 2+ ) present in the reaction and is also modulated by the nascent RNA structure and the trigger loop in the RNAP active site. Our data reveal remarkable plasticity of the RNAP active site in response to various regulatory stimuli and highlight functional diversity of transcription factors that bind inside the secondary channel of RNAP.RNA polymerase | transcriptional pausing | Gfh factors | Deinococcus radiodurans | stress response C ellular RNA polymerases (RNAPs) are complex molecular machines whose activity during transcription is regulated by DNA-and RNA-encoded signals, protein factors, small molecules, and inhibitors. The catalytic cycle of RNAP can be interrupted by pauses of various natures that play important roles in genetic regulation in all organisms, from the classic systems of transcription attenuation and their variations in bacteria (1) to recently discovered widespread promoter-proximal pausing in eukaryotes (2). The pausing serves to activate or repress transcription rapidly at specific genomic sites in response to regulatory stimuli and to coordinate RNA synthesis with other genetic processes (e.g., DNA replication and repair, RNA translation in bacteria) (1,(3)(4)(5)(6)(7)(8).Recent structural and biochemical studies revealed distinct RNAP conformations corresponding to different functional states of the transcription elongation complex (TEC) during nucleotide addition, RNA proofreading, and pausing (9-11). The control of structural transitions between these states likely underlies the function of multiple regulatory factors acting on RNAP. However, the roles of individual RNAP conformations in transcription and the mechanisms of their switching by transcription factors remain only partially understood. During transcription, RNAP holds the DNA template and the RNA transcript within its main channel, whose opening is controlled by the mobile clamp/shelf module and the flap domain of RNAP (9-11). Nucleotide addition and TEC translocation depend on alternating cycles of the folding of the trigger loop (TL) and kinking of the bridge helix (BH) in the RNAP active site (9-11) (Fig. 1A). Analysis of the stru...

Gfh factors and NusA cooperate to stimulate transcriptional pausing and termination

“…On the whole, our data demonstrate that Gfh factors act on a variety of paused transcription complexes, including elemental, hairpin‐dependent, post‐translocated, backtracking‐dependent, and consensus pauses, which significantly differ in their structures and translocation registers of the RNAP active site. At the same time, Dra Gfh factors have little effect on the rate of nucleotide addition in nonpaused catalytically active TECs . Previous studies suggested that Gfh factors recognize and stabilize a specific ratcheted conformation of RNAP .…”

“…). As our previous study suggested that Gfh factors do not act on backtracked TECs (in particular, they cannot compete with GreA in such complexes) , Gfh1 probably stimulates the initial step of pausing preceding RNAP backtracking .…”

“…We tested the effects of Dra Gfh1 (which has stronger effects on transcription than Gfh2, ) and NusA factors on different types of transcriptional pauses including (Fig. S1): (a) pretranslocated hairpin‐dependent hisP pause; (b) elemental pause ; (c) post‐translocated T7A1P pause ; and (d) backtracking‐dependent pause that constitutes a part of σ‐dependent pause signal found in the initially transcribed region of the lac UV5 promoter ( lac UV5P) .…”

“…We first analyzed the hisP pause, which can be stimulated by NusA in the case of E. coli RNAP and is highly sensitive to Gfh factors in the case of Dra RNAP . As the activity of Dra Gfh factors was shown to be dependent on the presence of manganese ions , which are greatly accumulated in Dra cells under stress conditions , the reactions were performed in the presence of either Mg 2+ or Mn 2+ ions. The DNA template contained the hisP sequence fused to the λP R promoter, resulting in pausing at position +77 (Fig.…”

“…Thus, strong pause stimulation requires a combination of changes imposed on the TEC by both factors, suggesting that they act through different pathways rather than different steps in the same pathway. In particular, Gfh factors likely stabilize the ratcheted RNAP conformation and induce binding of an additional Mn 2+ ion in the RNAP active site , while NusA might affect the translocation state of the TEC . Interestingly, Mn 2+ ions decrease NusA effects on the hisP pause, while simultaneously stimulating Gfh1 action, suggesting that they may somehow change TEC conformation during pausing.…”