Initially transcribed sequences strongly affect the extent of abortive initiation by RNA polymerase II

Abstract

We investigated transcript initiation and early elongation by RNA polymerase II using templates mismatched between -9 and +3 (bubble templates). Highly purified RNA polymerase II alone was able to initiate transcription specifically on these templates in the presence of dinucleotide primers. The length distribution of abortively initiated RNAs was similar for purified RNA polymerase II on bubble templates and polymerase II on double- stranded templates in HeLa nuclear extracts. Increasing the U content in the initial portion of the transcript caused similar increases in abortive initiation for transcription of bubble templates by pure polymerase and double-stranded templates in extracts. Thus, the level of abortive initiation by RNA polymerase II is at least partly determined by interactions of the polymerase with the transcript and/or the template, independent of the general transcription factors. Substitution of 5-bromo-UTP for UTP reduced abortive initiation on bubble templates, consistent with the idea that transcription complex stability during early elongation depends on the strength of the initial RNA-DNA hybrid. Interestingly, transcription of bubble templates in HeLa extracts gave very high levels of abortive initiation, suggesting that inability to reanneal the initially melted template segment inhibits transcript elongation in the presence of the initiation factors.