A Two-Way Street: Regulatory Interplay between RNA Polymerase and Nascent RNA Structure

Zhang, Jinwei; Landick, Robert

doi:10.1016/j.tibs.2015.12.009

Cited by 121 publications

(136 citation statements)

References 139 publications

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“…The GreB factor has likely evolved to specifically activate RNA cleavage in such extensively backtracked TECs, but its existence is limited to E. coli and related bacteria (40,41). Thus, RNAP stalling at genomic pause sites or long backtracking events, which make it resistant to both intrinsic and Gre-dependent RNA cleavage, might have important consequences for transcription regulation, resolution of the transcription-replication conflicts, and coordination of transcription with translation (42)(43)(44).…”

Section: Discussionmentioning

confidence: 99%

Conserved functions of the trigger loop and Gre factors in RNA cleavage by bacterial RNA polymerases

Miropolskaya

Esyunina

Kulbachinskiy

2017

Journal of Biological Chemistry

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RNA cleavage by RNA polymerase (RNAP) is the central step in co-transcriptional RNA proofreading. Bacterial RNAPs were proposed to rely on the same mobile element of the active site, the trigger loop (TL), for both nucleotide addition and RNA cleavage. RNA cleavage can also be stimulated by universal Gre factors, which should replace the TL to get access to the RNAP active site. The contributions of the TL and Gre factors to RNA cleavage reportedly vary between RNAPs from different bacterial species and, probably, different types of transcription complexes. Here, by comparing RNAPs from Escherichia coli, Deinococcus radiodurans, and Thermus aquaticus, we show that the functions of the TL and Gre factors in RNA cleavage are conserved in various species, with important variations that may be related to extremophilic adaptation. Deletions of the TL strongly impair intrinsic RNA cleavage by all three RNAPs and eliminate the interspecies differences in the reaction rates. GreA factors activate RNA cleavage by wild-type RNAPs to similar levels. The rates of GreA-dependent cleavage are lower for ⌬TL RNAP variants, suggesting that the TL contributes to the Gre function. Finally, neither the TL nor GreA can efficiently activate RNA cleavage in certain types of backtracked transcription complexes, suggesting that these complexes adopt a catalytically inactive conformation probably important for transcription regulation.The RNA cleavage reaction is thought to play a crucial role in correction of transcriptional errors during RNA synthesis by RNAP 2 (1-4). Because this reaction is performed by the same active site of RNAP as nucleotide addition, RNAP must "backtrack" along the DNA template after nucleotide misincorporation to allow cleavage of internal phosphodiester bonds in RNA. Long backtracking events can result in permanent stalling of RNAP in the backtracked state, and RNA cleavage also serves for reactivation of such "arrested" transcription elongation complexes (TECs) (5-7). After backtracking, the RNA 3Ј-end is accommodated within the secondary RNAP channel, which normally serves for NTP entry (Fig. 1). Both nucleotide addition and RNA cleavage require two divalent metal ions (usually Mg 2ϩ ) bound in the RNAP active site, which coordinate the reaction substrates (3,8). In addition to metal ions, several other factors were shown to contribute to RNA cleavage. In particular, the reaction is greatly stimulated by RNA nucleotide at position ϩ2 relative to the active site through its direct interactions with the second metal ion and the attacking water molecule (1). The ϩ2 nucleotide was also proposed to stimulate the second metal ion binding by changing the geometry of the active site residues involved in metal chelation ("active site tuning") (3). Recent crystallographic analysis of a bacterial backtracked TEC revealed that the ϩ2 RNA nucleotide is accommodated in an evolutionary conserved RNAP cavity ("proofreading site"), which facilitates catalysis (Fig. 1, left) (9). Similarly, interactions of the ϩ2 nucleotide i...

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Section: Discussionmentioning

confidence: 99%

Conserved functions of the trigger loop and Gre factors in RNA cleavage by bacterial RNA polymerases

Miropolskaya

Esyunina

Kulbachinskiy

2017

Journal of Biological Chemistry

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“…Nascent RNA can also induce pausing by forming either secondary structures or R-loops that interfere with the transcription elongation complex. For example, RNA hairpins that form within or upstream of the RNA exit channel cause pausing [30,72]. R-loops are also implicated in Pol II pausing during transcription termination [73,74].…”

Section: Causes Of Rna Polymerase Pausingmentioning

confidence: 99%

Pause & go: from the discovery of RNA polymerase pausing to its functional implications

Mayer

Landry

Churchman

2017

Current Opinion in Cell Biology

122

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The synthesis of nascent RNA is a discontinuous process in which phases of productive elongation by RNA polymerase are interrupted by frequent pauses. Transcriptional pausing was first observed decades ago, but was long considered to be a special feature of transcription at certain genes. This view was challenged when studies using genome-wide approaches revealed that RNA polymerase II pauses at promoter-proximal regions in large sets of genes in Drosophila and mammalian cells. High-resolution genomic methods uncovered that pausing is not restricted to promoters, but occurs globally throughout gene-body regions, implying the existence of key-rate limiting steps in nascent RNA synthesis downstream of transcription initiation. Here, we outline the experimental breakthroughs that led to the discovery of pervasive transcriptional pausing, discuss its emerging roles and regulation, and highlight the importance of pausing in human development and disease.

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“…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).…”

mentioning

confidence: 99%

Regulation of transcriptional pausing through the secondary channel of RNA polymerase

Esyunina

Agapov

Kulbachinskiy

2016

Proc. Natl. Acad. Sci. U.S.A.

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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...

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A Two-Way Street: Regulatory Interplay between RNA Polymerase and Nascent RNA Structure

Cited by 121 publications

References 139 publications

Conserved functions of the trigger loop and Gre factors in RNA cleavage by bacterial RNA polymerases

Conserved functions of the trigger loop and Gre factors in RNA cleavage by bacterial RNA polymerases

Pause & go: from the discovery of RNA polymerase pausing to its functional implications

Regulation of transcriptional pausing through the secondary channel of RNA polymerase

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