Promoter escape by RNA polymerase II. Downstream promoter DNA is required during multiple steps of early transcription

Abstract

Recent evidence, obtained in a reconstituted RNA polymerase II transcription system, indicated that the promoter escape stage of transcription requires template DNA located downstream of the elongating polymerase. In the absence of downstream DNA, very early elongation complexes are unable to synthesize transcripts longer than approximately 10-14 nucleotides. In contrast, once transcripts longer than approximately 15 nucleotides have been synthesized, an extended region of downstream DNA is no longer required (Dvir, A., Tan, S., Conaway, J. W., and Conaway, R. C. (1997) J. Biol. Chem. 272, 28175-28178). In this work, we sought to define precisely when, during the synthesis of the first 10-15 phosphodiester bonds, downstream DNA is required. We report that, for complete promoter escape, downstream DNA extending to position 40/42 is required. The polymerase can be forced to arrest at several points prior to the completion of promoter escape by removing downstream DNA proximally to positions 40/42. The positions at which the polymerase arrests appear to be determined by the length of available downstream DNA, with arrest occurring at a relatively fixed position of approximately 28 nucleotides to the distal end of the template. A similar requirement is observed for transcription initiation, i.e. the formation of the first phosphodiester bond of nascent transcripts. In addition, we show that the requirement for a downstream region is independent of downstream DNA sequence, suggesting that the requirement reflects a general mechanism. Taken together, our results indicate (i) that downstream DNA is required continuously through the synthesis of the first 14-15 phosphodiester bonds of nascent transcripts, and (ii) that a major conformational change in the transcription complex likely occurs only after the completion of promoter escape.