Fluorescent Affinity Labeling of Initiation Site on Ribonucleic Acid Polymerase of Escherichia Coli

Felicia Y.H. Wu, Cheng Wen Wu

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39 Scopus citations


A fluorescent analog of 6-methylthioinosinedicarboxaldehyde (MMPR-OP) has been synthesized in which the methyl group is replaced by N-(acetylaminoethyl)-1-naphthylamine-5-sulfonate. This fluorescent nucleotide analog (AMPR-OP) is a much more potent inhibitor of DNA dependent RNA polymerase of Escherichia coli than MMPROP. The concentration of AMPR-OP required to inhibit 50 % of RNA polymerase activity is 7 × 10-6 m as compared to 5 × 10-4 m for MMPR-OP. The noncompetitive inhibition of AMPR-OP with respect to nucleoside triphosphate suggests that AMPR-OP binds to a site on the enzyme involved in the initiation of RNA chains. The inhibition of DNA dependent [32P]PP1 exchange reaction by low concentrations of AMPROP further support the contention that this compound primarily inhibits the initiation of RNA chains. When RNA polymerase was incubated with excess AMPR-OP followed by NaBH4 reduction, the dye was stoichiometrically bound to the enzyme. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the denatured, labeled enzyme indicates that AMPR-OP is covalently attached to the β subunit of the enzyme. Although the labeled enzyme is essentially inactive, fluorescence studies show that it still retains the ability to bind DNA template and nucleoside triphosphates. The binding of nucleoside triphosphates is presumably to the second nucleotide site (the polymerization site) on the enzyme and its specificity is dependent on the template. Furthermore, upon binding of the template and nucleoside triphosphates, the enzyme undergoes conformational changes. Energy transfer measurements indicate that the initiation site and rifampicin binding site are at least 37 Å apart. Thus the inhibitory effects of rifampicin on initiation of RNA chains is indirectly mediated through enzyme molecule.

Original languageEnglish
Pages (from-to)2562-2566
Number of pages5
Issue number12
StatePublished - 1 Jun 1974


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