Intrinsic transcript cleavage activity of RNA polymerase.

Orlova, Marianna; Newlands, Janet T.; Das, Asis; Goldfarb, Alex; Borukhov, Sergei

doi:10.1073/pnas.92.10.4596

Cited by 200 publications

(198 citation statements)

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“…At first glance, the ss DNA-ss RNA nuclease activity of T7 RNAP appears to be somewhat different from the known GreB-enhanced intrinsic cleavage activity of E. coli RNAP. The GreB-enhanced E. coli RNAP cleaves the nascent RNA in arrested/paused ternary complexes into fragments larger than T7 RNAP (24,25). The T7 RNAP nuclease pattern resembles that of GreA-enhanced E. coli RNAP, eucaryotic pol II, or vaccinia RNAP in that these enzymes seem to cut in mono-or dinucleotide steps (23,61).…”

Section: Models For Cleavage and Unusual Polymerization During Elongamentioning

confidence: 99%

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Nuclease Activity of T7 RNA Polymerase and the Heterogeneity of Transcription Elongation Complexes

Sastry

Ross

1997

Journal of Biological Chemistry

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We have discovered that T7 RNA polymerase, purified to apparent homogeneity from overexpressing Escherichia coli cells, possesses a DNase and an RNase activity. Mutations in the active center of T7 RNA polymerase abolished or greatly decreased the nuclease activity. This nuclease activity is specific for single-stranded DNA and RNA oligonucleotides and does not manifest on double-stranded DNAs. Under the conditions of promoter-driven transcription on double-stranded DNA, no nuclease activity was observed. The nuclease attacks DNA oligonucleotides in mono-or dinucleotide steps. The nuclease is a 3 to 5 exonuclease leaving a 3 -OH end, and it degrades DNA oligonucleotides to a minimum size of 3 to 5 nucleotides. It is completely dependent on Mg 2؉ . The T7 RNA polymerase-nuclease is inhibited by T7 lysozyme and heparin, although not completely. In the presence of rNTPs, the nuclease activity is suppressed but an unusual 3 -end-initiated polymerase activity is unmasked. RNA from isolated preelongation and elongation complexes arrested by a psoralen roadblock or naturally paused at the 3 -end of an oligonucleotide template exhibited evidence of nuclease activity. The nuclease activity of T7 RNA polymerase is unrelated to pyrophosphorolysis. We propose that the nuclease of T7 RNA polymerase acts only in arrested or paused elongation complexes, and that in combination with the unusual 3 -end polymerizing activity, causes heterogeneity in elongation complexes. Additionally, during normal transcription elongation, the kinetic balance between nuclease and polymerase is shifted in favor of polymerase.Transcription by procaryotic DNA-dependent RNA polymerases can be represented as follows (Scheme I).Elongation Termination SCHEME I. Representation of a transcription cycle. Open complexes synthesize short RNAs (up to 10 nts) during abortive initiation. After clearing the promoter, RNAP enters the elongation phase. Termination occurs at certain DNA sequences in either a factor-dependent or a factor-independent manner (2). The current view of transcription elongation has been possible because of our ability to arrest elongation at specific sites on DNA templates, partial purification of arrested complexes, and the enzymatic and chemical probing of their structures (3-17).The unexpected discovery of an RNA cleavage reaction in arrested Escherichia coli complexes (18) was followed by documentation of analogous cleavage reactions in other RNAPs (19 -23). E. coli RNAP and eucaryotic RNAP II are capable of RNA cleavage in binary and ternary complexes (24 -26). GreA and GreB of E. coli enhance the intrinsic cleavage by E. coli RNAP (25,27,28). GreA and GreB (and a eucaryotic counterpart, SII) prevent elongation arrest (29). GreA may participate in the fidelity of RNA synthesis (30).T7 RNAP (98.8 kDa) belongs to a class of single-subunit RNAPs that includes T3 and SP6 phage RNAPs (31-33). The three-dimensional structure of T7 RNAP shows high ␣-helicity with a deep cleft. Using a novel photochemical cross-linking technique, we have id...

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Section: Models For Cleavage and Unusual Polymerization During Elongamentioning

confidence: 99%

“…E. coli RNAP and eucaryotic RNAP II are capable of RNA cleavage in binary and ternary complexes (24 -26). GreA and GreB of E. coli enhance the intrinsic cleavage by E. coli RNAP (25,27,28). GreA and GreB (and a eucaryotic counterpart, SII) prevent elongation arrest (29).…”

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Nuclease Activity of T7 RNA Polymerase and the Heterogeneity of Transcription Elongation Complexes

Sastry

Ross

1997

Journal of Biological Chemistry

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“…RNAP was purified according to Burgess and Jendrisak (1975) from E. coli strain AD8571, which carries disrupted greA and greB genes (Orlova et al 1995). GreB protein was a generous gift from Dr. S. Borukhov (State University of New York Health Science Center, Brooklyn).…”

Section: Methodsmentioning

confidence: 99%

The Nun protein of bacteriophage HK022 inhibits translocation of Escherichia coliRNA polymerase without abolishing its catalytic activities

Hung¹,

Gottesman²

1997

Genes Dev.

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Bacteriophage HK022 Nun protein blocks transcription elongation by Escherichia coli RNA polymerase in vitro without dissociating the transcription complex. Nun is active on complexes located at any template site tested. Ultimately, only the 3 -OH terminal nucleotide of the nascent transcript in an arrested complex can turn over; it is removed by pyrophosphate and restored with NTPs. This suggests that Nun inhibits the translocation of RNA polymerase without abolishing its catalytic activities. Unlike spontaneously arrested complexes, Nun-arrested complexes cannot be reactivated by transcription factor GreB. The various complexes show distinct patterns of nucleotide incorporation and pyrophosphorolysis before or after treatment with Nun, suggesting that the configuration of RNAP, transcript, and template DNA is different in each complex.

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“…First, RNA polymerase III complexes catalyze transcript cleavage, but a separate cleavage-stimulating factor has not been reported for RNA polymerase III. Second, ternary complexes of Escherichia coli RNA polymerase purified from a greA Ϫ greB Ϫ strain retain a low level of transcript cleavage activity (20), an activity stimulated by basic pH. Third, mammalian RNA polymerase II pyrophosphorylizes its transcript to give products identical to those induced by treatment with TFIIS (21).…”

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Intrinsic Transcript Cleavage in Yeast RNA Polymerase II Elongation Complexes

Weilbaecher

Awrey

Edwards

et al. 2003

Journal of Biological Chemistry

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Transcript elongation can be interrupted by a variety of obstacles, including certain DNA sequences, DNAbinding proteins, chromatin, and DNA lesions. Bypass of many of these impediments is facilitated by elongation factor TFIIS through a mechanism that involves cleavage of the nascent transcript by the RNA polymerase II/TFIIS elongation complex. Highly purified yeast RNA polymerase II is able to perform transcript hydrolysis in the absence of TFIIS. The "intrinsic" cleavage activity is greatly stimulated at mildly basic pH and requires divalent cations. Both arrested and stalled complexes can carry out the intrinsic cleavage reaction, although not all stalled complexes are equally efficient at this reaction. Arrested complexes in which the nascent transcript was cleaved in the absence of TFIIS were reactivated to readthrough blocks to elongation. Thus, cleavage of the nascent transcript is sufficient for reactivating some arrested complexes. Small RNA products released following transcript cleavage in stalled ternary complexes differ depending upon whether the cleavage has been induced by TFIIS or has occurred in mildly alkaline conditions. In contrast, both intrinsic and TFIIS-induced small RNA cleavage products are very similar when produced from an arrested ternary complex. Although ␣-amanitin interferes with the transcript cleavage stimulated by TFIIS, it has little effect on the intrinsic cleavage reaction. A mutant RNA polymerase previously shown to be refractory to TFIIS-induced transcript cleavage is essentially identical to the wild type polymerase in all tested aspects of intrinsic cleavage.Gene expression can be controlled by regulating any step of the transcription cycle: promoter binding, initiation, promoter escape, elongation, or termination. After transcript synthesis begins, the ability to suppress or complete the synthesis of an RNA transcript is vital to the cell, and a substantial and growing number of genes have been reported to be regulated during transcript elongation (1).The ternary elongation complex consists of the RNA polymerase, the template DNA, and the nascent RNA transcript. Surratt et al. (2) discovered that bacterial RNA polymerase ternary complexes have a mechanism for transcript shortening, endonucleolytic cleavage near the 3Ј-end of the transcript. After transcript cleavage, the 3Ј-fragment is released while the 5Ј-fragment is retained in an active ternary complex. A single cleavage event can liberate from 1 to 17 nt 1 of RNA bearing a 5Ј-monophosphate (3-6). Remarkably, transcription accurately resumes from the site of transcript cleavage to re-synthesize the excised RNA.Transcript cleavage activity is conserved in many DNA-dependent RNA polymerases, including bacterial RNA polymerases, vaccinia virus RNA polymerase (7), RNA polymerase I (8, 9), RNA polymerase II (10 -14), and RNA polymerase III (15). Accessory factors that stimulate transcript cleavage in ternary complexes have been identified in both prokaryotes (GreA and GreB) and eukaryotes (reviewed in Refs. 3,9,[16][17]...

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Intrinsic transcript cleavage activity of RNA polymerase.

Abstract: ABSTRACT

Cited by 200 publications

References 22 publications

Nuclease Activity of T7 RNA Polymerase and the Heterogeneity of Transcription Elongation Complexes

Nuclease Activity of T7 RNA Polymerase and the Heterogeneity of Transcription Elongation Complexes

The Nun protein of bacteriophage HK022 inhibits translocation of Escherichia coliRNA polymerase without abolishing its catalytic activities

Intrinsic Transcript Cleavage in Yeast RNA Polymerase II Elongation Complexes

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