Electrophoretic separation of polyadenylation-specific complexes.

Abstract

A polyadenylation-specific complex composed of precursor RNA containing the adenovirus-2 L3 site and HeLa cellular components was detected by electrophoresis on a native, low-percentage polyacrylamide gel. Upon incubation in a reaction containing ATP and nuclear extract, precursor RNA was rapidly assembled into this complex. This assembly did not require poly(A) synthesis, as it occurred efficiently in the presence of ATP analogs that inhibited this reaction. Mutation of the hexanucleotide AAUAAA 20 nucleotides upstream of the L3 site to AAGAAA or deletion of sequence between +5 and +48 nucleotides downstream of the L3 site inactivates polyadenylation. The specific complex did not effectively from on substrate RNA with either the AAGAAA mutation or the downstream deletion mutation. Kinetic experiments showed that the assembly of this complex preceded processing of precursor RNA. We proposed that formation of this complex represents an intermediate step in polyadenylation.