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Role of Sulfhydryl Residues of Escherichia coli Ribonucleic Acid Polymerase in Template Recognition and Specific Initiation

From the ¹ From the Department of Biophysics, Albert Einstein College of Medicine, Bronx, New York 10461

Sulfhydryl residues in RNA polymerase of Escherichia coli have been reversibly modified by treatment with a thiol reagent, sodium tetrathionate. After extensive reaction periods, 21 sulfhydryl residues of the holoenzyme are modified. Such enzyme preparations do not catalyze templatedependent or -independent synthesis of poly(A-U) or DNA-dependent pyrophosphate exchange reaction. Interactions of the enzyme with nucleoside triphosphates, d(A-T) copolymer, and an inhibitor, rifampicin, are greatly altered, while the binding of another inhibitor, Rose Bengal, is unaffected. Analytical ultracentrifugation of this extensively reacted enzyme shows no disintegration into subunits. However, fluorescence studies with a noncovalent, environmentally-sensitive probe, p-toluidinyl-napthalene-6-sulfonate, reveal that the modified enzyme undergoes conformational alterations as a result of reaction of the sulfhydryl residues. Treatment of holoenzyme with tetrathionate for short time periods produces an enzyme preparation containing an average of 12 modified sulfhydryl residues. This enzyme preparation is almost inactive (<5% initial activity) when assayed with native double-stranded DNA templates; however, it retains 50 to 70% of the initial activity with d(A-T) copolymer, and 70 to 100% of the initial activity with single-stranded DNA templates. The sigma subunit isolated from this modified holoenzyme preparation is fully active, whereas the core enzyme isolated from such preparations shows only slight stimulation by addition of the unmodified sigma subunit. Thus, the main structural defect responsible for the inability of the modified holoenzyme to transcribe DNA resides on the core enzyme. Although sigma subunit may be needed, our results indicate that intact core enzyme is essential for template recognition and specific initiation.

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