Synthesis of a cleavable dinucleotide photoaffinity probe of ribonucleic acid polymerase: Application to trinucleotide labeling of an Escherichia coli transcription complex

Michelle M. Hanna, Claude F. Meares

Research output: Contribution to journalArticle

30 Citations (Scopus)

Abstract

The cleavable dinucleotide photoaffinity probe 5′-[[(4-azidophenacyl)thio]phosphoryl]adenylyl(3′-5′)uridine was prepared and used to determine the 5′ contacts of a trinucleotide in an Escherichia coli RNA polymerase/T7 DNA transcription complex. The probe was prepared by alkylating 5′-(thiophosphory)adenylyl(3′-5′)uridine with azidophenacyl bromide. The 5′-(thiophosphoryl)adenylyl(3′-5′)uridine was prepared by the abortive initiation reaction of RNA polymerase on a poly[d(A-T)] DNA template, using adenosine 5′-O-(thiomonophosphate) and uridine triphosphate as substrates. A transcription complex containing a radiolabeled trinucleotide at the Al promoter of bacteriophage T7 D111 or D123 DNA was prepared by using the dinucleotide photoaffinity probe as initiator and cytidine [α-32P]triphosphate as the other substrate. After photolysis, the labeled subunits and DNA were isolated, and the trinucleotide was removed in the presence of phenylmercuric acetate and analyzed by polyacrylamide gel electrophoresis. The 5′ end of the trinucleotide was found to label the DNA (≈88%) and also the β (≈10%) and σ(≈3%) subunits of E. coli RNA polymerase. It was also shown that the order of migration of the β and β′ subunits of E. coli RNA polymerase on polyacrylamide gel electrophoresis in sodium dodecyl sulfate is different from that in sodium dodecyl sulfate plus urea.

Original languageEnglish (US)
Pages (from-to)3546-3551
Number of pages6
JournalBiochemistry
Volume22
Issue number15
StatePublished - 1983

Fingerprint

Transcription
Labeling
Escherichia coli
Uridine
DNA-Directed RNA Polymerases
RNA
DNA
Electrophoresis
Sodium Dodecyl Sulfate
Polyacrylamide Gel Electrophoresis
Phenylmercuric Acetate
Bacteriophage T7
Cytidine Triphosphate
Cytidine
Uridine Triphosphate
Bacteriophages
Photolysis
Substrates
Bromides
Urea

ASJC Scopus subject areas

  • Biochemistry

Cite this

Synthesis of a cleavable dinucleotide photoaffinity probe of ribonucleic acid polymerase : Application to trinucleotide labeling of an Escherichia coli transcription complex. / Hanna, Michelle M.; Meares, Claude F.

In: Biochemistry, Vol. 22, No. 15, 1983, p. 3546-3551.

Research output: Contribution to journalArticle

@article{e55fb719d4314b08a4beae58290545a1,
title = "Synthesis of a cleavable dinucleotide photoaffinity probe of ribonucleic acid polymerase: Application to trinucleotide labeling of an Escherichia coli transcription complex",
abstract = "The cleavable dinucleotide photoaffinity probe 5′-[[(4-azidophenacyl)thio]phosphoryl]adenylyl(3′-5′)uridine was prepared and used to determine the 5′ contacts of a trinucleotide in an Escherichia coli RNA polymerase/T7 DNA transcription complex. The probe was prepared by alkylating 5′-(thiophosphory)adenylyl(3′-5′)uridine with azidophenacyl bromide. The 5′-(thiophosphoryl)adenylyl(3′-5′)uridine was prepared by the abortive initiation reaction of RNA polymerase on a poly[d(A-T)] DNA template, using adenosine 5′-O-(thiomonophosphate) and uridine triphosphate as substrates. A transcription complex containing a radiolabeled trinucleotide at the Al promoter of bacteriophage T7 D111 or D123 DNA was prepared by using the dinucleotide photoaffinity probe as initiator and cytidine [α-32P]triphosphate as the other substrate. After photolysis, the labeled subunits and DNA were isolated, and the trinucleotide was removed in the presence of phenylmercuric acetate and analyzed by polyacrylamide gel electrophoresis. The 5′ end of the trinucleotide was found to label the DNA (≈88{\%}) and also the β (≈10{\%}) and σ(≈3{\%}) subunits of E. coli RNA polymerase. It was also shown that the order of migration of the β and β′ subunits of E. coli RNA polymerase on polyacrylamide gel electrophoresis in sodium dodecyl sulfate is different from that in sodium dodecyl sulfate plus urea.",
author = "Hanna, {Michelle M.} and Meares, {Claude F.}",
year = "1983",
language = "English (US)",
volume = "22",
pages = "3546--3551",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "15",

}

TY - JOUR

T1 - Synthesis of a cleavable dinucleotide photoaffinity probe of ribonucleic acid polymerase

T2 - Application to trinucleotide labeling of an Escherichia coli transcription complex

AU - Hanna, Michelle M.

AU - Meares, Claude F.

PY - 1983

Y1 - 1983

N2 - The cleavable dinucleotide photoaffinity probe 5′-[[(4-azidophenacyl)thio]phosphoryl]adenylyl(3′-5′)uridine was prepared and used to determine the 5′ contacts of a trinucleotide in an Escherichia coli RNA polymerase/T7 DNA transcription complex. The probe was prepared by alkylating 5′-(thiophosphory)adenylyl(3′-5′)uridine with azidophenacyl bromide. The 5′-(thiophosphoryl)adenylyl(3′-5′)uridine was prepared by the abortive initiation reaction of RNA polymerase on a poly[d(A-T)] DNA template, using adenosine 5′-O-(thiomonophosphate) and uridine triphosphate as substrates. A transcription complex containing a radiolabeled trinucleotide at the Al promoter of bacteriophage T7 D111 or D123 DNA was prepared by using the dinucleotide photoaffinity probe as initiator and cytidine [α-32P]triphosphate as the other substrate. After photolysis, the labeled subunits and DNA were isolated, and the trinucleotide was removed in the presence of phenylmercuric acetate and analyzed by polyacrylamide gel electrophoresis. The 5′ end of the trinucleotide was found to label the DNA (≈88%) and also the β (≈10%) and σ(≈3%) subunits of E. coli RNA polymerase. It was also shown that the order of migration of the β and β′ subunits of E. coli RNA polymerase on polyacrylamide gel electrophoresis in sodium dodecyl sulfate is different from that in sodium dodecyl sulfate plus urea.

AB - The cleavable dinucleotide photoaffinity probe 5′-[[(4-azidophenacyl)thio]phosphoryl]adenylyl(3′-5′)uridine was prepared and used to determine the 5′ contacts of a trinucleotide in an Escherichia coli RNA polymerase/T7 DNA transcription complex. The probe was prepared by alkylating 5′-(thiophosphory)adenylyl(3′-5′)uridine with azidophenacyl bromide. The 5′-(thiophosphoryl)adenylyl(3′-5′)uridine was prepared by the abortive initiation reaction of RNA polymerase on a poly[d(A-T)] DNA template, using adenosine 5′-O-(thiomonophosphate) and uridine triphosphate as substrates. A transcription complex containing a radiolabeled trinucleotide at the Al promoter of bacteriophage T7 D111 or D123 DNA was prepared by using the dinucleotide photoaffinity probe as initiator and cytidine [α-32P]triphosphate as the other substrate. After photolysis, the labeled subunits and DNA were isolated, and the trinucleotide was removed in the presence of phenylmercuric acetate and analyzed by polyacrylamide gel electrophoresis. The 5′ end of the trinucleotide was found to label the DNA (≈88%) and also the β (≈10%) and σ(≈3%) subunits of E. coli RNA polymerase. It was also shown that the order of migration of the β and β′ subunits of E. coli RNA polymerase on polyacrylamide gel electrophoresis in sodium dodecyl sulfate is different from that in sodium dodecyl sulfate plus urea.

UR - http://www.scopus.com/inward/record.url?scp=0021107920&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0021107920&partnerID=8YFLogxK

M3 - Article

C2 - 6351906

AN - SCOPUS:0021107920

VL - 22

SP - 3546

EP - 3551

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 15

ER -