Accessibility of the leading end of ribonucleic acid in transcription complexes

Susan L. Bernhard, Claude F. Meares

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

A photoaffinity-protection technique has been developed to study the accessibility of the leading (5′) end of nascent RNA as it passes through the transcription complex formed by Escherichia coli RNA polymerase and phage T7 DNA. The macromolecules contacted by the leading (5′) end of the growing RNA chain in the transcription complex have been determined previously by photoaffinity labeling experiments using aryl azides attached to the leading end of nascent RNA [Hanna, M. M., & Meares, C. F. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 4238-4242]. By using thiols to reduce accessible photoprobes, we have modified the photoaffinity technique so that it tests the accessibility of the leading end of nascent RNA to small molecules in solution, as a function of RNA chain length. We examined in detail RNA molecules containing 11-50 nucleotides, whose 5′ ends label the β and β′ enzyme subunits with good yield. The thiol's accessibility to the leading end of each transcript was determined by comparing the RNAs cross-linked to ββ′ in thiol-treated samples to controls not treated with thiol. Incubation with 1 mM dithiothreitol for 5 min reduced approximately 36% of the 5′-azides on RNAs 11-13 bases long and approximately 43% on RNAs 28-37 bases long but practically none of the 5′-azides on RNAs 40-43 bases long. Also notable was the reduction of 34 ± 1% of the 5′-azides on RNA 12 bases long but only 14 ± 2% on the 14-base RNA; on the T7 A1 promoter, the leading end of the transcript diverges from the DNA template when the chain is between 12 and 14 bases long.

Original languageEnglish (US)
Pages (from-to)6397-6404
Number of pages8
JournalBiochemistry
Volume25
Issue number21
StatePublished - 1986

Fingerprint

Transcription
RNA
Azides
Sulfhydryl Compounds
RNA Phages
Coliphages
Bacteriophage T7
Molecules
Bacteriophages
Dithiothreitol
DNA
DNA-Directed RNA Polymerases
Macromolecules
Chain length
Labeling
Escherichia coli
Labels
Nucleotides

ASJC Scopus subject areas

  • Biochemistry

Cite this

Bernhard, S. L., & Meares, C. F. (1986). Accessibility of the leading end of ribonucleic acid in transcription complexes. Biochemistry, 25(21), 6397-6404.

Accessibility of the leading end of ribonucleic acid in transcription complexes. / Bernhard, Susan L.; Meares, Claude F.

In: Biochemistry, Vol. 25, No. 21, 1986, p. 6397-6404.

Research output: Contribution to journalArticle

Bernhard, SL & Meares, CF 1986, 'Accessibility of the leading end of ribonucleic acid in transcription complexes', Biochemistry, vol. 25, no. 21, pp. 6397-6404.
Bernhard, Susan L. ; Meares, Claude F. / Accessibility of the leading end of ribonucleic acid in transcription complexes. In: Biochemistry. 1986 ; Vol. 25, No. 21. pp. 6397-6404.
@article{89025dad66784265a5b1abf85cccb47e,
title = "Accessibility of the leading end of ribonucleic acid in transcription complexes",
abstract = "A photoaffinity-protection technique has been developed to study the accessibility of the leading (5′) end of nascent RNA as it passes through the transcription complex formed by Escherichia coli RNA polymerase and phage T7 DNA. The macromolecules contacted by the leading (5′) end of the growing RNA chain in the transcription complex have been determined previously by photoaffinity labeling experiments using aryl azides attached to the leading end of nascent RNA [Hanna, M. M., & Meares, C. F. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 4238-4242]. By using thiols to reduce accessible photoprobes, we have modified the photoaffinity technique so that it tests the accessibility of the leading end of nascent RNA to small molecules in solution, as a function of RNA chain length. We examined in detail RNA molecules containing 11-50 nucleotides, whose 5′ ends label the β and β′ enzyme subunits with good yield. The thiol's accessibility to the leading end of each transcript was determined by comparing the RNAs cross-linked to ββ′ in thiol-treated samples to controls not treated with thiol. Incubation with 1 mM dithiothreitol for 5 min reduced approximately 36{\%} of the 5′-azides on RNAs 11-13 bases long and approximately 43{\%} on RNAs 28-37 bases long but practically none of the 5′-azides on RNAs 40-43 bases long. Also notable was the reduction of 34 ± 1{\%} of the 5′-azides on RNA 12 bases long but only 14 ± 2{\%} on the 14-base RNA; on the T7 A1 promoter, the leading end of the transcript diverges from the DNA template when the chain is between 12 and 14 bases long.",
author = "Bernhard, {Susan L.} and Meares, {Claude F.}",
year = "1986",
language = "English (US)",
volume = "25",
pages = "6397--6404",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "21",

}

TY - JOUR

T1 - Accessibility of the leading end of ribonucleic acid in transcription complexes

AU - Bernhard, Susan L.

AU - Meares, Claude F.

PY - 1986

Y1 - 1986

N2 - A photoaffinity-protection technique has been developed to study the accessibility of the leading (5′) end of nascent RNA as it passes through the transcription complex formed by Escherichia coli RNA polymerase and phage T7 DNA. The macromolecules contacted by the leading (5′) end of the growing RNA chain in the transcription complex have been determined previously by photoaffinity labeling experiments using aryl azides attached to the leading end of nascent RNA [Hanna, M. M., & Meares, C. F. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 4238-4242]. By using thiols to reduce accessible photoprobes, we have modified the photoaffinity technique so that it tests the accessibility of the leading end of nascent RNA to small molecules in solution, as a function of RNA chain length. We examined in detail RNA molecules containing 11-50 nucleotides, whose 5′ ends label the β and β′ enzyme subunits with good yield. The thiol's accessibility to the leading end of each transcript was determined by comparing the RNAs cross-linked to ββ′ in thiol-treated samples to controls not treated with thiol. Incubation with 1 mM dithiothreitol for 5 min reduced approximately 36% of the 5′-azides on RNAs 11-13 bases long and approximately 43% on RNAs 28-37 bases long but practically none of the 5′-azides on RNAs 40-43 bases long. Also notable was the reduction of 34 ± 1% of the 5′-azides on RNA 12 bases long but only 14 ± 2% on the 14-base RNA; on the T7 A1 promoter, the leading end of the transcript diverges from the DNA template when the chain is between 12 and 14 bases long.

AB - A photoaffinity-protection technique has been developed to study the accessibility of the leading (5′) end of nascent RNA as it passes through the transcription complex formed by Escherichia coli RNA polymerase and phage T7 DNA. The macromolecules contacted by the leading (5′) end of the growing RNA chain in the transcription complex have been determined previously by photoaffinity labeling experiments using aryl azides attached to the leading end of nascent RNA [Hanna, M. M., & Meares, C. F. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 4238-4242]. By using thiols to reduce accessible photoprobes, we have modified the photoaffinity technique so that it tests the accessibility of the leading end of nascent RNA to small molecules in solution, as a function of RNA chain length. We examined in detail RNA molecules containing 11-50 nucleotides, whose 5′ ends label the β and β′ enzyme subunits with good yield. The thiol's accessibility to the leading end of each transcript was determined by comparing the RNAs cross-linked to ββ′ in thiol-treated samples to controls not treated with thiol. Incubation with 1 mM dithiothreitol for 5 min reduced approximately 36% of the 5′-azides on RNAs 11-13 bases long and approximately 43% on RNAs 28-37 bases long but practically none of the 5′-azides on RNAs 40-43 bases long. Also notable was the reduction of 34 ± 1% of the 5′-azides on RNA 12 bases long but only 14 ± 2% on the 14-base RNA; on the T7 A1 promoter, the leading end of the transcript diverges from the DNA template when the chain is between 12 and 14 bases long.

UR - http://www.scopus.com/inward/record.url?scp=0023003042&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0023003042&partnerID=8YFLogxK

M3 - Article

C2 - 2431709

AN - SCOPUS:0023003042

VL - 25

SP - 6397

EP - 6404

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 21

ER -