Transcription arrest caused by long nascent RNA chains

Thomas Bentin; Dmitry Cherny; H Jakob Larsen; Peter E Nielsen

doi:10.1016/j.bbaexp.2004.12.006

Transcription arrest caused by long nascent RNA chains

Thomas Bentin, Dmitry Cherny, H Jakob Larsen, Peter E Nielsen

Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › peer review

16 Citationer (Scopus)

Abstract

Udgivelsesdato: 2005-Feb-14

Originalsprog	Engelsk
Tidsskrift	BBA General Subjects
Vol/bind	1727
Udgave nummer	2
Sider (fra-til)	97-105
Antal sider	8
ISSN	0304-4165
DOI	https://doi.org/10.1016/j.bbaexp.2004.12.006
Status	Udgivet - 2004

Bibliografisk note

Keywords: DNA; Kinetics; Nucleic Acid Conformation; RNA; Ribonuclease H; Templates, Genetic; Transcription, Genetic

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10.1016/j.bbaexp.2004.12.006

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Citationsformater

@article{9456c4d0eea211ddbf70000ea68e967b,

title = "Transcription arrest caused by long nascent RNA chains",

abstract = "The transcription process is highly processive. However, specific sequence elements encoded in the nascent RNA may signal transcription pausing and/or termination. We find that under certain conditions nascent RNA chains can have a strong and apparently sequence-independent inhibitory effect on transcription. Using phage T3 RNA polymerase (T3 RNAP) and covalently closed circular (cccDNA) DNA templates that did not contain any strong termination signal, transcription was severely inhibited after a short period of time. Less than approximately 10% residual transcriptional activity remained after 10 min of incubation. The addition of RNase A almost fully restored transcription in a dose dependent manner. Throughout RNase A rescue, an elongation rate of approximately 170 nt/s was maintained and this velocity was independent of RNA transcript length, at least up to 6 kb. Instead, RNase A rescue increased the number of active elongation complexes. Thus transcription behaved as an all-or-none process. The mechanism of transcription inhibition was explored using electron microscopy and further biochemical experiments. The data suggest that multiple mechanisms may contribute to the observed effects. Part of the inhibition can be ascribed to the formation of R-loops between the nascent RNA and the DNA template, which provides {"}roadblocks{"} to trailing T3 RNAPs. Based on available literature we discuss possible in vivo implications of the results.",

author = "Thomas Bentin and Dmitry Cherny and Larsen, {H Jakob} and Nielsen, {Peter E}",

note = "Keywords: DNA; Kinetics; Nucleic Acid Conformation; RNA; Ribonuclease H; Templates, Genetic; Transcription, Genetic",

year = "2004",

doi = "10.1016/j.bbaexp.2004.12.006",

language = "English",

volume = "1727",

pages = "97--105",

journal = "BBA General Subjects",

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TY - JOUR

T1 - Transcription arrest caused by long nascent RNA chains

AU - Bentin, Thomas

AU - Cherny, Dmitry

AU - Larsen, H Jakob

AU - Nielsen, Peter E

N1 - Keywords: DNA; Kinetics; Nucleic Acid Conformation; RNA; Ribonuclease H; Templates, Genetic; Transcription, Genetic

PY - 2004

Y1 - 2004

N2 - The transcription process is highly processive. However, specific sequence elements encoded in the nascent RNA may signal transcription pausing and/or termination. We find that under certain conditions nascent RNA chains can have a strong and apparently sequence-independent inhibitory effect on transcription. Using phage T3 RNA polymerase (T3 RNAP) and covalently closed circular (cccDNA) DNA templates that did not contain any strong termination signal, transcription was severely inhibited after a short period of time. Less than approximately 10% residual transcriptional activity remained after 10 min of incubation. The addition of RNase A almost fully restored transcription in a dose dependent manner. Throughout RNase A rescue, an elongation rate of approximately 170 nt/s was maintained and this velocity was independent of RNA transcript length, at least up to 6 kb. Instead, RNase A rescue increased the number of active elongation complexes. Thus transcription behaved as an all-or-none process. The mechanism of transcription inhibition was explored using electron microscopy and further biochemical experiments. The data suggest that multiple mechanisms may contribute to the observed effects. Part of the inhibition can be ascribed to the formation of R-loops between the nascent RNA and the DNA template, which provides "roadblocks" to trailing T3 RNAPs. Based on available literature we discuss possible in vivo implications of the results.

AB - The transcription process is highly processive. However, specific sequence elements encoded in the nascent RNA may signal transcription pausing and/or termination. We find that under certain conditions nascent RNA chains can have a strong and apparently sequence-independent inhibitory effect on transcription. Using phage T3 RNA polymerase (T3 RNAP) and covalently closed circular (cccDNA) DNA templates that did not contain any strong termination signal, transcription was severely inhibited after a short period of time. Less than approximately 10% residual transcriptional activity remained after 10 min of incubation. The addition of RNase A almost fully restored transcription in a dose dependent manner. Throughout RNase A rescue, an elongation rate of approximately 170 nt/s was maintained and this velocity was independent of RNA transcript length, at least up to 6 kb. Instead, RNase A rescue increased the number of active elongation complexes. Thus transcription behaved as an all-or-none process. The mechanism of transcription inhibition was explored using electron microscopy and further biochemical experiments. The data suggest that multiple mechanisms may contribute to the observed effects. Part of the inhibition can be ascribed to the formation of R-loops between the nascent RNA and the DNA template, which provides "roadblocks" to trailing T3 RNAPs. Based on available literature we discuss possible in vivo implications of the results.

U2 - 10.1016/j.bbaexp.2004.12.006

DO - 10.1016/j.bbaexp.2004.12.006

M3 - Journal article

C2 - 15716026

SN - 0304-4165

VL - 1727

SP - 97

EP - 105

JO - BBA General Subjects

JF - BBA General Subjects

IS - 2

ER -