Coupled transcription-polyadenylation in a cell-free system

To investigate the relationship between transcription and polyadenylation, an in vitro system has been developed in which endogenously transcribed pre-mRNAs containing functional polyadenylation sites are rapidly and accurately cleaved in a HeLa nuclear extract. Cleavage of endogenously transcribed substrates differed from that of exogenous substrates in that a proximal 3' terminus was not required, the reaction was more tolerant of increased Mg2+ levels, and endogenous substrates were cleaved more efficiently. A promoter dependence for this reaction was suggested by the observation that substrates transcribed by bacteriophage T7 RNA polymerase in the presence of nuclear extract were not cleaved. In addition, analysis of the bovine growth hormone poly(A) site indicated that it is highly efficient in vitro which agrees with previous in vivo data. The availability of an in vitro system in which transcription and polyadenylation are coupled should facilitate analysis of the relation between 3' end processing and RNA polymerase II transcription termination as well as the promoter requirements for polyadenylation.