Intrinsic RNA hairpin-mediated transcription termination at high temperature in Thermus aquaticus

ABSTRACT Transcription termination establishes gene boundaries and limits regulatory interference. In bacteria, intrinsic termination, mediated by an RNA hairpin followed by a polyuridine tract (polyU), is a principal mechanism. Because RNA secondary structures are destabilized at high temperature, whether intrinsic termination functions effectively in thermophiles has been uncertain. We mapped RNA 3′ termini genome-wide in the extreme thermophile Thermus aquaticus ( Taq ) grown at 70 °C using Term-Seq. Computational analysis identified hairpins upstream of most termini. These hairpins were substantially more thermally stable than those of the mesophile Escherichia coli . RNA 3′ ends were also detected within coding regions, consistent with transcriptional pausing or premature termination at spurious hairpins in GC-rich transcripts. In vitro transcription showed that native Taq terminators direct efficient intrinsic termination by Taq RNA polymerase at 70 °C and require both hairpin and polyU. Termination remained efficient up to 80 °C. At this temperature, the terminator hairpin RNA is unfolded in solution, indicating that RNA polymerase promotes hairpin formation during transcription. Thus, intrinsic termination operates efficiently in thermophiles. At lower temperatures, expression of the Rho termination factor increased, suggesting that factor-dependent termination may contribute more prominently under cooler growth conditions in Thermus . Significance Statement Intrinsic transcription termination requires formation of an RNA hairpin. Hairpin structures are expected to be unstable at the high temperatures inhabited by thermophilic bacteria. Whether and how hairpin-dependent termination operates under these conditions has remained unknown. Using genome-wide transcripts ends mapping and biochemical approaches, we show that the RNA polymerase of Thermus aquaticus promotes folding of the nascent RNA into a terminator hairpin. This enables efficient termination even at extreme temperatures. Raising temperature favours entropy-driven dissociation of the transcription complex. At lower temperatures, termination appears to increasingly rely on a factor-dependent pathway. Together, these results identify RNA polymerase as an active participant in hairpin-dependent termination and show how temperature may shift the balance between alternative termination mechanisms.