Spectroscopic determination of open complex formation at promoters for Escherichia coli RNA polymerase

Jennifer J. Sullivan, Keith P. Bjornson, Lawrence Sowers, Pieter L. DeHaseth

Research output: Contribution to journalArticle

28 Citations (Scopus)

Abstract

A considerable amount of effort has been expended studying the kinetics of association of Escherichia coli RNA polymerase with promoter DNA in forming the open complex. Strand separation occurs over about 12 base pairs and includes the transcription start site. However, these efforts have been significantly hampered by the lack of a sensitive, real time method by which formation of an open complex could be assayed. Here, we employ short (86 bp) synthetic promoters with 2-aminopurine (2-AP) substitutions in the region that becomes single-stranded to spectroscopically monitor open complex formation. We demonstrate that promoters bearing the substitutions behave in a manner similar to that of those containing only the four common bases with respect to both the region of strand separation and start site selection. Open complex formation was found to yield an increased fluorescence signal with an emission maximum characteristic of 2-aminopurine. This spectroscopic assay for open complex formation was found to be well-suited to the investigation of a strong promoter, allowing open complex formation to be followed over a time scale of seconds with a stopped flow apparatus. The introduction of two additional nonconsensus base pairs in the -35 region resulted in a promoter for which open complex formation was 100-fold slower. The same substrates were also used to monitor the promoter re-annealing that ensues upon initiation of RNA synthesis. Similar rates for this process were observed for the two promoter variants employed in this study.

Original languageEnglish (US)
Pages (from-to)8005-8012
Number of pages8
JournalBiochemistry
Volume36
Issue number26
DOIs
StatePublished - Jul 1 1997
Externally publishedYes

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2-Aminopurine
DNA-Directed RNA Polymerases
Base Pairing
Escherichia coli
Substitution reactions
Bearings (structural)
Site selection
Transcription Initiation Site
Assays
Fluorescence
Association reactions
RNA
Annealing
Kinetics
DNA
Substrates

ASJC Scopus subject areas

  • Biochemistry

Cite this

Spectroscopic determination of open complex formation at promoters for Escherichia coli RNA polymerase. / Sullivan, Jennifer J.; Bjornson, Keith P.; Sowers, Lawrence; DeHaseth, Pieter L.

In: Biochemistry, Vol. 36, No. 26, 01.07.1997, p. 8005-8012.

Research output: Contribution to journalArticle

Sullivan, Jennifer J. ; Bjornson, Keith P. ; Sowers, Lawrence ; DeHaseth, Pieter L. / Spectroscopic determination of open complex formation at promoters for Escherichia coli RNA polymerase. In: Biochemistry. 1997 ; Vol. 36, No. 26. pp. 8005-8012.
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