Lineage-specific variations in the trigger loop modulate RNA proofreading by bacterial RNA polymerases

Esyunina, Daria; Turtola, Matti; Pupov, Danil; Bass, I. A.; Klimašauskas, Saulius; Belogurov, Georgiy A.; Kulbachinskiy, Andrey

doi:10.1093/nar/gkv1521

Cited by 27 publications

(37 citation statements)

References 66 publications

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“…1B). Mn 2+ ions, by themselves, may also affect the active site structure and the TL conformation (27,42,43), thus making RNAP more sensitive to the Gfh action and increasing its affinity to Gfh.…”

Section: Discussionmentioning

confidence: 99%

“…We cloned and purified Gfh factors from Dra and analyzed their effects on Dra RNAP activity at different steps of transcription. All experiments were performed in the presence of either Mg 2+ or Mn 2+ ions as RNAP cofactors, because manganese ions were previously shown to play essential roles in stress response in Dra (23)(24)(25)(26) and to modulate the activity of Dra RNAP (27).…”

Section: Manganese-dependent Effects Of Gfh Factors On Transcriptionmentioning

confidence: 99%

“…The experiments were performed with the minimal nucleic scaffold template that is bound by RNAP in the posttranslocated state and was previously used extensively to characterize catalysis by Dra RNAP (27)(28)(29) (Fig. S3A).…”

Section: Manganese-dependent Effects Of Gfh Factors On Transcriptionmentioning

confidence: 99%

“…GreA greatly increased the RNA cleavage rate by Dra RNAP in the same TEC (27) (Fig. S3 C and D); we therefore performed the cleavage reactions not only at pH 7.9 but also at pH 6.5, to decrease the reaction rate and make the measurements more accurate.…”

Section: Manganese-dependent Effects Of Gfh Factors On Transcriptionmentioning

confidence: 99%

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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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Section: Discussionmentioning

confidence: 99%

Section: Manganese-dependent Effects Of Gfh Factors On Transcriptionmentioning

confidence: 99%

Section: Manganese-dependent Effects Of Gfh Factors On Transcriptionmentioning

confidence: 99%

Section: Manganese-dependent Effects Of Gfh Factors On Transcriptionmentioning

confidence: 99%

See 2 more Smart Citations

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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“…In the paused TEC structure, the central part of the TL is disordered, and the ultimate RNA phosphodiester bond is shifted upstream of the active center (28). Functions of the trigger loop and GreA in RNA cleavage temperatures as EcoRNAP, displays much higher RNA cleavage rates than the latter (24). In this work we directly compared RNA cleavage by these three RNAPs at various temperatures and in TECs of different structures.…”

mentioning

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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Gfh factors and NusA cooperate to stimulate transcriptional pausing and termination

et al. 2017

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Lineage-specific Gfh factors from the radioresistant bacterium Deinococcus radiodurans, which bind within the secondary channel of RNA polymerase, stimulate transcriptional pausing at a wide range of pause signals (elemental, hairpin-dependent, post-translocated, backtracking-dependent, and consensus pauses) and increase intrinsic termination. Universal bacterial factor NusA, which binds near the RNA exit channel, enhances the effects of Gfh factors on termination and hairpin-dependent pausing but do not act on other pause sites. It is proposed that NusA and Gfh target different steps in the pausing pathway and may act together to regulate transcription under stress conditions. Thus, transcription factors that interact with nascent RNA in the RNA exit channel can communicate with secondary channel regulators to modulate RNA polymerase activities.

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Lineage-specific variations in the trigger loop modulate RNA proofreading by bacterial RNA polymerases

Cited by 27 publications

References 66 publications

Regulation of transcriptional pausing through the secondary channel of RNA polymerase

Regulation of transcriptional pausing through the secondary channel of RNA polymerase

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

Gfh factors and NusA cooperate to stimulate transcriptional pausing and termination

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