GreA and GreB proteins revive backtracked RNA polymerase in vivo by promoting transcript trimming

Toulme, Francine; Mosrin-Huaman, Christine; Sparkowski, Jason; Das, Asis; Leng, Marc; Rahmouni, A. Rachid

doi:10.1093/emboj/19.24.6853

Cited by 95 publications

(99 citation statements)

References 38 publications

(51 reference statements)

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“…However, after the replicase has synthesized more than 12 nucleotides of the nascent RNA, it is more stably associated with the template RNA (41,45). It is not known whether viral RNA replicases can escape from a stalled ternary complex, as had been demonstrated for DNA-dependent RNA polymerases (46). These observations could be adapted to study the ternary complexes of RNA-dependent RNA polymerases.…”

Section: Discussionmentioning

confidence: 95%

Pokeweed Antiviral Protein Inhibits Brome Mosaic Virus Replication in Plant Cells

Picard

Kao

Hudak

2005

Journal of Biological Chemistry

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Pokeweed antiviral protein (PAP) is a ribosome-inactivating protein isolated from the pokeweed plant (Phytolacca americana) that inhibits the proliferation of several plant and animal viruses. We have shown previously that PAP and nontoxic mutants of PAP can directly depurinate brome mosaic virus (BMV) RNA in vitro, resulting in reduced viral protein translation. Here we expand on these initial studies and, using a barley protoplast system, demonstrate that recombinant PAP and nontoxic mutants isolated from E. coli are able to reduce the accumulation of BMV RNAs in vivo. Pretreatment of only BMV RNA3 with PAP prior to transfection of barley protoplasts reduced the accumulation of all BMV RNAs, with a more severe effect on subgenomic RNA4 levels. Using in vitro RNA synthesis assays, we show that a depurinated template causes the BMV replicase to stall at the template nucleotide adjacent to the missing base. These results provide new insight into the antiviral mechanism of PAP, namely that PAP depurination of BMV RNA impedes both RNA replication and subgenomic RNA transcription. These novel activities are distinct from the PAP-induced reduction of viral RNA translation and represent new targets for the inhibition of viral infection. Pokeweed antiviral protein (PAP)1 is a 29-kDa ribosomeinactivating protein of the pokeweed plant Phytolacca americana. Since its initial description as an antiviral agent against tobacco mosaic virus (1), PAP has been demonstrated to reduce the propagation of several plant and animal viruses, including potato virus X, HIV, and influenza (2-4). It therefore holds promise as a broad-spectrum antiviral agent.Years after its initial discovery, the enzymatic activity of PAP was characterized as an N-glycosylase (5). Like all ribosome-inactivating proteins, PAP efficiently removes a conserved adenine from the sarcin/ricin loop within domain VI of the large ribosomal RNA (6, 7). This depurination slows the elongation step of protein synthesis and is considered to be the reason for cytotoxicity of the protein (reviewed in Refs. 8 and 9).The accompanying decline in cellular protein translation may cause local cell death and limit virus propagation (10). This model is supported by observations showing a positive correlation between ribosome depurination and inhibition of virus infection (11). The accompanying decline in cellular protein translation, as a result of depurination, is often cited as the cause of antiviral activity. For example, reduction of poliovirus infection of HeLa cells incubated with PAP was attributed to inhibition of translation in virus-infected cells (12). In addition, inhibition of tobacco mosaic virus multiplication in tobacco protoplasts correlated well with PAP-mediated inhibition of translation (13).More recent results have revealed that many ribosome-inactivating proteins are capable of depurinating RNA substrates apart from the rRNA (14 -16). Rajamohan et al. (17) showed that PAP removes both adenines and guanines from HIV-1 when incubated in vitro with the genomic...

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Section: Discussionmentioning

confidence: 95%

Pokeweed Antiviral Protein Inhibits Brome Mosaic Virus Replication in Plant Cells

Picard

Kao

Hudak

2005

Journal of Biological Chemistry

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“…The backtracked RNA can be cleaved either by the intrinsic endonucleolytic activity of RNAP (very inefficient at physiological pH) or by the action of extrinsic transcript cleavage factors GreA and GreB (TFIIS in eukaryotic RNA polymerase II system (42)). This cleavage leads to realignment of the newly formed RNA 3′-terminus with the active site and reactivation of transcription (pause release) (43)(44)(45). Due to these parallels, we wanted to examine if backtracking and pausing occur in transcription initiation by RNAP as well.…”

Section: Significancementioning

confidence: 99%

“…It is well established that elongating RNAP can backtrack and pause (44,45). In such circumstances, the nascent RNA 3′ end backtranslocates into the secondary channel, where it can undergo endonucleolytic cleavage by the GreA-RNAP complex (25,44,48,66).…”

Section: Slower Transcription Initiation Kinetics From Select Ntp-stamentioning

confidence: 99%

Backtracked and paused transcription initiation intermediate of Escherichia coli RNA polymerase

Lerner

Chung

Allen

et al. 2016

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

111

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Initiation is a highly regulated, rate-limiting step in transcription. We used a series of approaches to examine the kinetics of RNA polymerase (RNAP) transcription initiation in greater detail. Quenched kinetics assays, in combination with gel-based assays, showed that RNAP exit kinetics from complexes stalled at later stages of initiation (e.g., from a 7-base transcript) were markedly slower than from earlier stages (e.g., from a 2-or 4-base transcript). In addition, the RNAP-GreA endonuclease accelerated transcription kinetics from otherwise delayed initiation states. Further examination with magnetic tweezers transcription experiments showed that RNAP adopted a long-lived backtracked state during initiation and that the pausedbacktracked initiation intermediate was populated abundantly at physiologically relevant nucleoside triphosphate (NTP) concentrations. The paused intermediate population was further increased when the NTP concentration was decreased and/or when an imbalance in NTP concentration was introduced (situations that mimic stress). Our results confirm the existence of a previously hypothesized paused and backtracked RNAP initiation intermediate and suggest it is biologically relevant; furthermore, such intermediates could be exploited for therapeutic purposes and may reflect a conserved state among paused, initiating eukaryotic RNA polymerase II enzymes.T ranscription in Escherichia coli comprises three stages: initiation, elongation and termination. Initiation, which is typically the rate-limiting and the most regulated stage of transcription, is by itself a complex, multistep process consisting of the following successive steps (1, 2): (i) association of RNA polymerase (RNAP) core enzyme (subunit composition α2ββ′ω) with the promoter specificity factor σ (such as σ70 for transcription of housekeeping genes) to form RNAP holoenzyme; (ii) binding of holoenzyme to the −10 and −35 DNA elements in the promoter recognition sequence (PRS) upstream to the transcription start site (TSS) to form closed promoter complex (RPc); (iii) isomerization of RPc through multiple intermediates into an open promoter complex (RPo), in which a ∼12-bp DNA stretch (bases at registers −10 to +2) is melted to form a transcription bubble, the template DNA strand is inserted into RNAP major cleft positioning the base at register +1 of TSS at the active site, the nontemplate strand is tightly bound by σ70 and the downstream DNA duplex (bases +3 up to +20) is loaded into RNAP β′ DNA-binding clamp; (iv) an abortive initiation step (AI), where binding of nucleoside triphosphate (NTPs) and the start of RNA synthesis leads to formation of an initial transcribing complex (RP ITC ) followed by RNAP cycling through multiple polymerization trials via a DNA scrunching mechanism (3, 4), release of short "abortive transcripts" and repositioning itself in RP O for a new synthesis trial (5-7); and finally, (v) RNAP promoter escape, when enough strain is built in the enzyme, the σ70 undergoes structural transition to relieve blockage of ...

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“…The cleavage reaction is intrinsic to the RNA polymerase itself, but is enhanced in the presence of accessory factors, such as TFIIS (Rudd et al 1994;Orlova et al 1995). The first example of transcript cleavage was provided for the Escherichia coli RNA polymerase under conditions that halted the RNA polymerase (Surratt et al 1991), and notably, the relief of backtracked RNA polymerase mediated by GreA and GreB was observed in vivo (Toulme et al 2000). A similar cleavage activity in arrested RNAP II occurs within active ternary complexes, and TFIIS is important for this activity (Izban and Luse 1992b;Reines 1992;Wang and Hawley 1993).…”

Section: Factors That Influence Transcriptional Arrest: Tfiismentioning

confidence: 99%

Elongation by RNA polymerase II: the short and long of it

Sims¹,

Belotserkovskaya²,

Reinberg³

2004

Genes Dev.

614

586

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Appreciable advances into the process of transcript elongation by RNA polymerase II (RNAP II) have identified this stage as a dynamic and highly regulated step of the transcription cycle. Here, we discuss the many factors that regulate the elongation stage of transcription. Our discussion includes the classical elongation factors that modulate the activity of RNAP II, and the more recently identified factors that facilitate elongation on chromatin templates. Additionally, we discuss the factors that associate with RNAP II, but do not modulate its catalytic activity. Elongation is highlighted as a central process that coordinates multiple stages in mRNA biogenesis and maturation.The regulation of gene expression is one of the most intensely studied areas in all of science. Differential gene expression in multicellular organisms forms the foundation of cell-type specificity. Deregulation of the appropriate pattern of gene expression has profound effects on cellular function and underlies many diseases. Although there are many cellular processes that control gene expression, the most direct regulation occurs during transcription. The transcription of protein-coding genes in eukaryotes is performed by RNA polymerase II (RNAP II). Up until recently, the vast majority of studies aimed at elucidating the molecular mechanisms of transcription regulation have focused on early stages, such as the formation of a transcription initiation complex (preinitiation) and initiation (see below). For many years, transcript elongation has been thought of as the trivial addition of ribonucleoside triphosphates to the growing mRNA chain. It is now apparent that this process is a dynamic and highly regulated stage of the transcription cycle capable of coordinating downstream events. Numerous factors have been identified that specifically target the elongation stage of transcription. Importantly, multiple steps in mRNA maturation, including premRNA capping, splicing, 3Ј-end processing, surveillance, and export, are modulated through interactions with the RNAP II transcript elongation complex (TEC). It also appears that distinct elongation factors function in specific transcriptional contexts; the requirements for these specialized factors are largely unknown and highlight the need to better understand elongation in vivo. Many molecular and biochemical approaches have been used to quantify the different aspects of elongation, many of which are discussed below. It remains important to assimilate both in vitro and in vivo experimental systems when discussing what constitutes an elongation factor. An elongation factor can be defined as any molecule that affects the activities of or is associated with the TEC. We suggest that there are at least two major types of transcript elongation factors: one family, referred to as active elongation factors, and a second family, denoted as passive elongation factors. The difference resides in whether the factor affects the catalytic activity of RNAP II, regardless of whether the factor remains associate...

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GreA and GreB proteins revive backtracked RNA polymerase in vivo by promoting transcript trimming

Cited by 95 publications

References 38 publications

Pokeweed Antiviral Protein Inhibits Brome Mosaic Virus Replication in Plant Cells

Pokeweed Antiviral Protein Inhibits Brome Mosaic Virus Replication in Plant Cells

Backtracked and paused transcription initiation intermediate of Escherichia coli RNA polymerase

Elongation by RNA polymerase II: the short and long of it

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