Antisense oligonucleotide-stimulated transcriptional pausing reveals RNA exit channel specificity of RNA polymerase and mechanistic contributions of NusA and RfaH.

Transcript elongation by bacterial RNA polymerase (RNAP) is thought to be regulated at pause sites by open versus closed positions of the RNAP clamp domain, pause-suppressing regulators like NusG and RfaH that stabilize the closed-clampRNAP conformation, and pause-enhancing regulators like NusA and exit channel nascent RNA structures that stabilize the open clamp RNAP conformation. However, the mutual effects of these protein and RNA regulators on RNAP conformation are incompletely understood. For example, it is unknown whether NusA directly interacts with exit channel duplexes and whether formation of exit channel duplexes and RfaH binding compete by favoring the open and closed RNAP conformations. We report new insights into these mechanisms using antisense oligonucleotide mimics of a pause RNA hairpin from the leader region of the his biosynthetic operon of enteric bacteria like Escherichia coli. By systematically varying the structure and length of the oligonucleotide mimic, we determined that full pause stabilization requires an RNA-RNA duplex of at least 8 bp or a DNA-RNA duplex of at least 11 bp; RNA-RNA duplexes were more effective than DNA-RNA. NusA stimulation of pausing was optimal with 10-bp RNA-RNA duplexes and was aided by single-stranded RNA upstream of the duplex but was significantly reduced with DNA-RNA duplexes. Our results favor direct NusA stabilization of exit channel duplexes, which consequently affect RNAP clamp conformation. Effects of RfaH, which suppresses oligo-stabilization of pausing, were competitive with antisense oligonucleotide concentration, suggesting that RfaH and exit channel duplexes compete via opposing effects on RNAP clamp conformation.