RNA polymerase II transcription terminates at a specific DNA sequence in a HeLa cell-free reaction

Recently, we identified a specific DNA sequence from the gastrin gene that regulates RNA polymerase II transcription termination in vivo. In the studies presented here, we examined the processing and termination activity of this sequence in vitro. When present in an in vitro synthesized RNA, this sequence (U9A2U5AU4AU4AU5) does not serve as an RNA processing signal on incubation with HeLa whole-cell extract. However, transcription of template DNA in HeLa whole-cell extract does terminate near the 5' end of this sequence. Nuclease S1 and exonuclease VII mapping of the 3' region of the in vitro synthesized RNAs confirm these results. The termination activity of the sequence A9T2A5TA4TA4TA5 is independent of the distance from the promoter and of the nature of the DNA template (linear vs. circular). The termination activity of the sequence shows a strong orientation dependence. These results strongly suggest that the termination activity of this cis-acting element is modulated by a trans-acting cellular factor. The unique structural feature of this sequence, a 10.5-base-pair inverted repeat, may determine the specificity of interaction of the trans-acting factor with the cis-acting element, resulting in accurate termination of transcription.