Transcription reinitiation by recycling RNA polymerase that diffuses on DNA after releasing terminated RNA

Abstract

Despite extensive studies on transcription mechanisms, it is unknown how termination complexes are disassembled, especially in what order the essential components dissociate. Our single-molecule fluorescence study unveils that RNA transcript release precedes RNA polymerase (RNAP) dissociation from the DNA template much more often than their concurrent dissociations in intrinsic termination of bacterial transcription. As termination is defined by the release of product RNA from the transcription complex, the subsequent retention of RNAP on DNA constitutes a previously unidentified stage, termed here as recycling. During the recycling stage, post-terminational RNAPs one-dimensionally diffuse on DNA in downward and upward directions, and can initiate transcription again at the original and nearby promoters in the case of retaining a sigma factor. The efficiency of this event, termed here as reinitiation, increases with supplement of a sigma factor. In summary, after releasing RNA product at intrinsic termination, recycling RNAP diffuses on the DNA template for reinitiation most of the time.

Alternative transcription cycle for bacterial RNA polymerase

“…Each elongation complex is assumed to be capable of stochastically selecting either the canonical or the alternative cycle at the time of termination. Many of our findings of RNAP initiation after termination are consistent with independent observations reported by Kang et al 46 , adding credence to the phenomena.…”

The δ subunit and NTPase HelD institute a two-pronged mechanism for RNA polymerase recycling

“…To further delineate the contributions of δ and HelD to nucleic acid displacement, we conducted band shift assays, in which we first bound RNAP to nucleic acids and subsequently added δ and/or HelD. We first tested displacement of DNA with an artificial bubble, which when bound to RNAP mimics a situation ensuing after many intrinsic termination events [3][4][5]40 . HelD displaced about 25% of DNA from RNAP ΔδΔHelD , while δ led to about 80% displacement in the absence of HelD (Fig.…”

“…At a terminator, bacterial EC is abruptly destabilized either by an oligo-U-tailed G/C-rich RNA hairpin or by the RNA translocase/helicase ρ 2 . However, RNAP can linger on DNA after RNA release 3 – 5 , roadblocking replisomes to trigger double-stranded DNA breaks 6 and giving rise to aberrant antisense transcripts 5 . RNAP can also form binary complexes with RNA 7 , 8 , either through de novo association with stable RNAs, such as tRNAs and 6S RNA 9 , 10 , or in the course of hairpin-induced termination 11 .…”

Hopping and Flipping of RNA Polymerase on DNA during Recycling for Reinitiation after Intrinsic Termination in Bacterial Transcription

“…Recently, on the other hand, two independent single-molecule fluorescence studies concurrently discovered 1D diffusion of post-terminational RNAP in E. coli transcription system [ 17 , 18 ]. After releasing RNA at intrinsic termination, RNAP mostly remains on DNA and one-dimensionally diffuses in both downstream and upstream directions, and recycles for reinitiation at nearby promoters.…”

“…This question is intriguing, particularly because the two observations on 1D diffusion of recycling RNAP are seemingly consistent with different mechanisms of diffusion, one observation with sliding and the other with hopping. One observation was that 1D diffusion of recycling RNAP is blocked by a roadblock bound on DNA [ 17 ]. This alludes the sliding mechanism, where RNAP does not leave DNA microscopically or temporarily, because the roadblock could interfere with sliding of protein along DNA [ 9 ] more efficiently than hopping [ 3 , 5 , 6 ].…”