Characterization of elongating T7 and SP6 RNA polymerases and their response to a roadblock generated by a site-specific DNA binding protein

Pavco, Pamela A.; Steege, Deborah A.

doi:10.1093/nar/19.17.4639

Cited by 48 publications

(45 citation statements)

References 43 publications

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“…Transcription elongation by phage RNA polymerases has been described to proceed with high efficiency through a variety of proteins bound to DNA (Wolffe et al, 1986;Pavco & Steege, 1991). The mechanism of RNA synthesis as well as the structure of the elongation complex are the subject of several recent studies (Erie et al, 1992;Johnson & Chamberlin, 1994;Altmann et al, 1994;Chamberlin, 1995;Maldonado & Reinberg, 1995;Nudler et al, 1995;Zhou et al, 1995), which have revealed the involvement of various structural, thermodynamic and kinetic factors of the different partners (DNA, RNA, RNA polymerase machinery).…”

Section: Introductionmentioning

confidence: 99%

Efficient Inhibition of Transcription Elongationin vitroby Oligonucleotide Phosphoramidates Targeted to Proviral HIV DNA

Giovannangéli

Perrouault

Escudé

et al. 1996

Journal of Molecular Biology

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Section: Introductionmentioning

confidence: 99%

Efficient Inhibition of Transcription Elongationin vitroby Oligonucleotide Phosphoramidates Targeted to Proviral HIV DNA

Giovannangéli

Perrouault

Escudé

et al. 1996

Journal of Molecular Biology

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“…In E. coli the activity of T7 RNAP may be affected by obstructing proteins, depending upon their location and the tightness of their binding (19,46). Thus, when bound at the very beginning of the transcribed sequence, the lac repressor causes T7 RNAP to terminate prematurely, whereas it has no effect when bound further downstream.…”

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confidence: 99%

The Histone-Like Protein HU Does Not Obstruct Movement of T7 RNA Polymerase in Escherichia coli Cells but Stimulates Its Activity

2002

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In vivo, RNA polymerases (RNAPs) do not transcribe naked DNA but do transcribe protein-associated DNA. Studies with the model enzyme T7 RNAP have shown that, in eukaryotic cells or in vitro, nucleosomes can inhibit both transcription initiation and elongation. We examine here whether the presence of HU, one of the major histone-like proteins in Escherichia coli cells (the genuine milieu for T7 RNAP) affects its activity. An engineered lac operon fused to the T7 late promoter was introduced into the chromosome of T7 RNAPproducing strains that either overexpress HU or lack it. The flows of RNAP that enter and exit this operon were compared with regard to the content of HU. We found that the fraction of T7 RNAP molecules that do not reach the end of the lac operon (ca. 15%) is the same whether the host cells overexpressed HU or lacked it: thus, the enzyme either freely displaces HU or transcribes through it. However, in these cells, the transcript yield was increased when HU is overexpressed and decreased in the hup mutants, presumably reflecting changes in DNA supercoiling. Thus, in contrast to eukaryotic nucleosomes, HU does not impair T7 RNAP activity but has a stimulatory effect. Finally, our results suggest that HU can also influence mRNA stability in vivo.Bacteriophage T7 RNA polymerase (RNAP), the prototype of T-odd phage-encoded RNAPs, has been the subject of considerable interest over the last 15 years. In vitro, this monomeric enzyme (99 kDa), which is much simpler than cellular RNAPs, can initiate transcription without the help of an additional factor(s) from a specific promoter (P T7 ) consisting of a highly conserved 21-nucleotide sequence. Its crystal structure has been determined both in isolation (56) and within the initiation complex (10). Virtually any DNA sequence can be efficiently transcribed when fused downstream of P T7 , a property which has proven invaluable for RNA studies. In addition, T7 RNAP can be expressed in a variety of hosts, including Escherichia coli, and this feature has been widely exploited for overexpressing genes of interest in these hosts (17,57,58).Because of its simplicity, T7 RNAP is also attractive for investigating fundamental aspects of transcription. In particular, it has been used as a model for studying the effect of histones on this process. In vitro, in a reconstituted system, the presence of a nucleosome core within the promoter region strongly inhibits initiation, whereas nucleosomes are less efficient in inhibiting elongation: readthrough transcription still occurs, but pausing and/or premature termination are enhanced (30, 43). More recently, it has been shown that incorporation of histone H1 into nucleosomes results in a much more drastic inhibition of both initiation and elongation (42). In vivo it was reported that transcription by T7 RNAP is inhibited by nucleosomes in Drosophila cells (38); similarly, in mammalian nuclei, the presence of a nearby enhancer can favor transcription initiation by T7 RNAP, but subsequent elongation is impaired (24).The natura...

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“…Elongation complexes are remarkably resilient in their elongating phase. T7 RNAP can transcribe past noncovalently bound proteins (38), DNA triple helices (39), and some base-specific adducts (40), whereas other roadblocks such as psoralens (3,4), acetoaminofluorine (40), and Z-DNA antibodies (41) can temporarily block elongation. RNA cleavage by bacteriophage T7-like RNAPs has not been demonstrated so far.…”

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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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Characterization of elongating T7 and SP6 RNA polymerases and their response to a roadblock generated by a site-specific DNA binding protein

Cited by 48 publications

References 43 publications

Efficient Inhibition of Transcription Elongationin vitroby Oligonucleotide Phosphoramidates Targeted to Proviral HIV DNA

Efficient Inhibition of Transcription Elongationin vitroby Oligonucleotide Phosphoramidates Targeted to Proviral HIV DNA

The Histone-Like Protein HU Does Not Obstruct Movement of T7 RNA Polymerase in Escherichia coli Cells but Stimulates Its Activity

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

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