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

Abstract

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...

Accounting for RNA polymerase heterogeneity reveals state switching and two distinct long-lived backtrack states escaping through cleavage

“…21,22,[29][30][31][32][33] Our results show further that backtrack-recovery is mediated by intrinsic RNA cleavage and not not by diffusional Brownian motion, indicating that a subset of paused TECs assume a conformational state in which intrinsic and GreBassisted RNA cleavage is hindered, delaying as such the escape to productive elongation. 25,34,40,[42][43][44][45][46] The availability of large datasets also allowed us to interrogate the sources of the poorly understood heterogeneity in transcription velocity and pause dynamics, providing support for previously postulated state-switching that we could link to stochastic alterations in the frequency of short pauses. 24,26,29,32,[47][48][49][50] We present a unified mechanistic model that integrates all key findings of previous biochemical and singlemolecule studies, and describes the origin and hierarchy of intrinsic pause states as framework for future studies.…”

“…39,47 Similarly, the lifetime of ~1 s for the frequent pause state P1 is compatible with the duration of the previously described elemental pause, and pause lifetimes compatible with our estimates of ~4 s (P2) and ~100 s (P3) have also been previously reported. 21,22,24,30,[32][33][34][35][36] Our large dataset (Table S2) and accompanying analysis procedure thus allow us to unify these three independently observed pause states in a single model.…”

“…21,22,[29][30][31][32][33] Besides hairpin-stabilized pauses, various biochemical studies have identified two distinct exponentially distributed backtracked transcriptional pause states with notably different lifetimes (~5 s vs. ~100 s). 21,22,24,28,[34][35][36][37] In contrast, the majority of single-molecule studies attributed all pauses exceeding 5 s to a single state with a broad power-law distribution of pause durations, which originate from diffusive TEC dynamics during RNAp backtracking. 15,23,36,[38][39][40] This discrepancy extends to our understanding of the recovery of backtrack-induced transcriptional pauses: while biochemical studies indicate that RNA cleavage is the dominant recovery pathway, several single-molecule and theoretical studies consider diffusional Brownian motion of the RNAp to be the main recovery pathway.…”

TFS and Spt4/5 accelerate transcription through archaeal histone‐based chromatin

“…Bacterial RNAP, archaeal RNAP and eukaryotic RNAP I/II/III all exhibit intrinsic endonuclease activity that can be stimulated by interactions with GreA and GreB (bacteria), TFS (archaea), TFIIS (also termed SII, eukarya -Pol II) (Hausner et al, 2000;Fish and Kane, 2002;Laptenko et al, 2003;Lange and Hausner, 2004;Miropolskaya et al, 2017). TFS and TFIIS are homologous, whereas GreA/GreB are analogous in function.…”

“…All cleavage stimulatory factors bind RNAP near the secondary channel and extend protein fingers toward the active center of RNAP. The tips of these protein extensions typically contain and donate acidic residues to the RNAP active center that stimulate endonuclease activity (Lange and Hausner, 2004;Symersky et al, 2006;Fouqueau et al, 2017;Miropolskaya et al, 2017). TFS interacts with RNAP through the secondary channel (Symersky et al, 2006) and primarily acts to restore catalytic activity to transcription elongation complexes (TECs) that have paused and subsequently reverse translocated to position an internal phosphodiester linkage in the bipartite active center of RNAP (Park et al, 2002;Symersky et al, 2006;Klein et al, 2011;Sekine et al, 2015;Lisica et al, 2016;Xu et al, 2017).…”