3' Processing and termination of mouse histone transcripts synthesized in vitro by RNA polymerase II

The highly expressed mouse histone H2a-614 gene is located 800 nt 5' of the histone H3-614 gene. There is a 140 nt sequence located 500 nt from the end of the H2-614 mRNA which has been defined as a transcription termination site for RNA polymerase II. We established an in vitro transcription system in which both 3' end processing and transcription termination occur. A template containing the adenovirus major late promoter, a portion of the histone H2a-614 coding region, its 3' processing signal, followed by the transcription termination site was transcribed in a nuclear extract prepared from mouse myeloma cells. Some of the transcripts synthesized in the extract were cleaved at the histone processing site in a reaction which was dependent both on the hairpin binding factor and the U7 snRNP. The efficiency of histone 3' end formation was similar both on synthetic transcripts and transcripts synthesized by RNA polymerase II. Defined transcripts, which were not processed and which mapped to the transcription termination site, were released from the template, suggesting that they were formed by transcription termination. Termination in vitro was dependent on a functional histone processing signal.