Different types of pausing modes during transcription initiation

ABSTRACT

In many cases, initiation is rate limiting to transcription. This due in part to the multiple cycles of abortive transcription that delay promoter escape and the transition from initiation to elongation. Pausing of transcription in initiation can further delay promoter escape. The previously hypothesized pausing in initiation was confirmed by two recent studies from Duchi et al.¹ and from Lerner, Chung et al.² In both studies, pausing is attributed to a lack of forward translocation of the nascent transcript during initiation. However, the two works report on different pausing mechanisms. Duchi et al. report on pausing that occurs during initiation predominantly on-pathway of transcript synthesis. Lerner, Chung et al. report on pausing during initiation as a result of RNAP backtracking, which is off-pathway to transcript synthesis. Here, we discuss these studies, together with additional experimental results from single-molecule FRET focusing on a specific distance within the transcription bubble. We show that the results of these studies are complementary to each other and are consistent with a model involving two types of pauses in initiation: a short-lived pause that occurs in the translocation of a 6-mer nascent transcript and a long-lived pause that occurs as a result of 1–2 nucleotide backtracking of a 7-mer transcript.

KEYWORDS:

• transcription initiation
• transcription pausing
• backtracking
• abortive initiation
• promoter escape
• single-molecule FRET

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Acknowledgments

We thank Dr. SangYoon Chung for fruitful discussions, Ms. Maya Segal for language editing and Mrs. Maya Lerner for preparation of illustrative vignettes of RNAP and promoter DNA, and also Prof. Richard Ebright for generously providing us with the triphosphate dinucleotide pppApA, as a gift. SW discloses equity in Nesher Technologies and intellectual property used in the research reported here. The work at UCLA was conducted in Dr. Weiss's Laboratory. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Funding

This work was supported by the NIH under grant numbers GM069709 (to SW) and GM095904 (to XM and SW); NSF under grant number MCB-1244175 (to SW).

Authors' contributions

EL and SW designed this study; EL developed, designed and performed the smFRET measurements and analyzed the data, in consultation with AI and X.M; JJL and SB prepared GreA protein; EL, SB, XM and SW wrote the point of view.