Real-time fluorescence assay system for gene transcription: simultaneous observation of protein/DNA binding, localized DNA melting, and mRNA production.
This article describes the development of an in vitro multicolor fluorescence assay system for studying protein/DNA complex formation, transcription bubble formation, and mRNA production. These studies were accomplished using three different fluorescent spectroscopic probes: rhodamine-labeled DNA (at the 5' position) to monitor protein/DNA complex formation, DNA internally labeled with the base analog 2-aminopurine in place of adenine to monitor transcription bubble formation, and gamma-fluorophore-labeled UTP nucleotide to measure mRNA transcription rates. Combining these three assay systems allows the simultaneous determination of protein/DNA binding, localized DNA melting transitions, and mRNA production at physiological concentrations of reagents (pM-nM) and millisecond timing resolution. The application of this multicolor fluorescence assay to Escherichia coli RNA polymerase reactions (binding, open complex formation, and mRNA production) demonstrates the importance of kinetically coupled events in gene transcription.
Dunkak
K S
KS
Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee, 37232, USA.
Otto
M R
MR
Beechem
J M
JM
D015075:2-Aminopurine / Q000737:chemistry; D001483:Base Sequence; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D005454:Fluorescence Polarization / Q000379:methods; D005456:Fluorescent Dyes; D007700:Kinetics; D009282:Naphthalenesulfonates; D011485:Protein Binding; D012333:RNA, Messenger / Q000096:biosynthesis; D012235:Rhodamines; D013050:Spectrometry, Fluorescence / Q000379:methods*; D014158:Transcription, Genetic
D017931:DNA Primers; D005456:Fluorescent Dyes; D009282:Naphthalenesulfonates; D012333:RNA, Messenger; D012235:Rhodamines; D015075:2-Aminopurine; C027081:UTP-1-aminonaphthalene-5-sulfonate; D004247:DNA; D012321:DNA-Directed RNA Polymerases