Interaction of a nascent RNA structure with RNA polymerase is required for hairpin-dependent transcriptional pausing but not for transcript release

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Abstract
Nascent RNA structures may regulate RNA chain elongation either directly through interaction with RNA polymerase or indirectly by disrupting nascent RNA contacts with polymerase or DNA. To distinguish these mechanisms we tested whether the effects of the hisleader pause RNA hairpin could be mimicked by pairing of antisense DNA or RNA oligonucleotides to the nascent transcript. The hispause hairpin inhibits nucleotide addition when it forms 11 nucleotides from the transcript 3′ end. It also can terminate transcription when base changes extend its stem to ≤8 nucleotides from the 3′ end. All oligonucleotides that disrupted the pause hairpin reduced the dwell time of RNA polymerase at the pause site dramatically, even when they mimicked the 11-nucleotide 3′-proximal RNA spacing or created a suitably positioned RNA loop. Oligonucleotides that paired ≤8 nucleotides from the pause RNA 3′ end could trigger transcript release, but only when added to an already paused complex. These results argue that direct interaction of a nascent RNA hairpin with RNA polymerase delays escape from a pause, but that indirect effects of a hairpin may trigger transcript release from a paused complex. Resistance of the paused complex to pyrophosphorolysis and its reversal by antisense oligonucleotides further suggest that interaction of the pause hairpin with RNA polymerase disengages the RNA 3′ end from the active site.