Probing yeast RNA polymerase A subunits with monospecific antibodies.

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Abstract
Monoclonal antibodies were raised in mouse against native RNA polymerase A from Saccharomyces cerevisiae. After screening with the spot-immunodetection technique, 14 hybridomas were selected and the antibodies produced in mice. Their specificity, analyzed by blot-immunodetection, was found to be marked biased towards a few RNA polymerase subunits: A135, A49, A43 and A14.5. A different monoclonal antibody directed against the larest subunit, A190, was obtained by immunizing a mouse with RNA polymerase A dissociated into its subunits with SDS [sodium dodecyl sulfate]. Two antibodies, which probably recognized the same antigenic determinant on subunit A135, inhibited in vitro RNA synthesis. Inhibition was prevented by preincubation of the enzyme with DNA, suggesting a role for the A135 subunit in template binding. The antibody directed against A14.5 interacted with the A14.5 kd [kilodalton] subunit present in all 3 forms of the yeast nuclear RNA polymerases but did not interfere with RNA polymerase activity. These antibody probes will be useful to study subunit function in reconstituted transcription systems.