Discontinuous Mechanism of Transcription Elongation

Nudler, Evgeny; Goldfarb, Alex; Kashlev, Mikhail

doi:10.1126/science.8047884

Cited by 142 publications

(176 citation statements)

References 11 publications

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“…Such complexes are particularly sensitive to endonucleolytic transcript cleavage stimulated by the E. coli GreB protein (Borukhov et al 1993). Transcript cleavage reactivates spontaneously arrested complexes, allowing elongation to continue from the new 3Ј-OH terminal nucleotide (Borukhov et al 1993;Nudler et al 1994). We asked if complexes arrested by Nun could also be reactivated by GreB.…”

Section: Nun-induced Transcription Arrest and Greb-stimulated Transcrmentioning

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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Section: Nun-induced Transcription Arrest and Greb-stimulated Transcrmentioning

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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“…For example, many helicases have unwinding rates which are independent of the NA sequence unwound [2,11]. Thus, the information content of the NA does not play an essential role in helicase operation-a large simplification compared to RNA polymerases, for example [12,13]. Monomeric helicases are a relatively simple system that may serve as a building block for future theoretical studies of helicases.…”

Section: Introductionmentioning

confidence: 99%

A Motor that Makes Its Own Track: Helicase Unwinding of DNA

Betterton

Jülicher

2003

Phys. Rev. Lett.

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Helicase opening of double-stranded nucleic acids may be "active" (the helicase directly destabilizes the dsNA to promote opening) or "passive" (the helicase binds ssNA available due to a thermal fluctuation which opens part of the dsNA). We describe helicase opening of dsNA, based on helicases which bind single NA strands and move towards the double-stranded region, using a discrete "hopping" model. The interaction between the helicase and the junction where the double strand opens is characterized by an interaction potential. The form of the potential determines whether the opening is active or passive. We calculate the rate of passive opening for the helicase PcrA, and show that the rate increases when the opening is active. Finally, we examine how to choose the interaction potential to optimize the rate of strand separation. One important result is our finding that active opening can increase the unwinding rate by 7 fold compared to passive opening.

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“…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)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17).…”

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confidence: 99%

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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Discontinuous Mechanism of Transcription Elongation

Cited by 142 publications

References 11 publications

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

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

A Motor that Makes Its Own Track: Helicase Unwinding of DNA

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

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