During DNA transcription, RNA polymerases often adopt inactive backtracked states. Recovery from backtracks can occur by 1D diffusion or cleavage of backtracked RNA, but how polymerases make this choice is unknown. Here, we use single-molecule optical tweezers experiments and stochastic theory to show that the choice of a backtrack recovery mechanism is determined by a kinetic competition between 1D diffusion and RNA cleavage. Notably, RNA polymerase I (Pol I) and Pol II recover from shallow backtracks by 1D diffusion, use RNA cleavage to recover from intermediary depths, and are unable to recover from extensive backtracks. Furthermore, Pol I and Pol II use distinct mechanisms to avoid nonrecoverable backtracking. Pol I is protected by its subunit A12.2, which decreases the rate of 1D diffusion and enables transcript cleavage up to 20 nt. In contrast, Pol II is fully protected through association with the cleavage stimulatory factor TFIIS, which enables rapid recovery from any depth by RNA cleavage. Taken together, we identify distinct backtrack recovery strategies of Pol I and Pol II, shedding light on the evolution of cellular functions of these key enzymes.Pol I | Pol II | transcription | optical tweezers | backtracking T ranscription in eukaryotes is catalyzed by three different RNA polymerases (Pol): Pol I, Pol II, and Pol III. These three enzymes share a common core and a highly conserved active site, but they vary in the number of subunits as well as the type of RNA that they produce (1). Pol I mainly produces ribosomal RNA (rRNA), Pol II makes messenger RNA (mRNA), and Pol III synthesizes mostly transfer RNA. Despite extensive research, many aspects of the micromechanical dynamics of transcription in eukaryotic polymerases remain unclear. During elongation, the polymerases move stepwise along a DNA template and produce complementary RNA. However, transcription elongation is not continuous, and it is often interrupted by polymerase backtracking, a reverse movement of RNA polymerase on the DNA template. This movement results in displacement of the RNA 3′ end from the active site and renders the enzyme transcriptionally inactive (2-6). Restarting transcription requires realigning the 3′ end of the RNA with the active site. The realignment can be achieved by either 1D diffusion of the enzyme along DNA (7-11) or endonucleolytic cleavage of the backtracked RNA (12-14) to generate a new 3′ end aligned with the active site. Importantly, eukaryotic RNA polymerases, Pol I and Pol II, differ in their RNA cleavage activities. Whereas Pol I has a strong cleavage activity that depends on the C-terminal domain of its subunit A12.2 (12, 15), Pol II has a weak intrinsic cleavage activity that requires its subunit Rpb9 and is strongly enhanced by the transcription factor TFIIS (16,17). Although 1D diffusion and RNA cleavage have been identified as mechanisms of backtrack recovery, it is not clear how the polymerases choose between these two different backtrack recovery strategies. Here, we used single-molecule optical twe...

Transcription is a key process in gene expression, in which RNA polymerases produce a complementary RNA copy from a DNA template. RNA polymerization is frequently interrupted by backtracking, a process in which polymerases perform a random walk along the DNA template. Recovery of polymerases from the transcriptionally-inactive backtracked state is determined by a kinetic competition between 1D diffusion and RNA cleavage. Here we describe backtrack recovery as a continuous-time random walk, where the time for a polymerase to recover from a backtrack of a given depth is described as a first-passage time of a random walker to reach an absorbing state. We represent RNA cleavage as a stochastic resetting process, and derive exact expressions for the recovery time distributions and mean recovery times from a given initial backtrack depth for both continuous and discrete-lattice descriptions of the random walk. We show that recovery time statistics do not depend on the discreteness of the DNA lattice when the rate of 1D diffusion is large compared to the rate of cleavage.

Throughout embryonic development and adult life, epithelia are subjected to compressive deformations. While these have been shown to trigger mechanosensitive responses such as cell extrusion and differentiation, which span tens of minutes, little is known about how epithelia adapt to compression over shorter timescales. Here, using suspended epithelia, we uncover the immediate response of epithelial tissues to the application of in-plane compressive strains (5-80%). We show that fast compression induces tissue buckling followed by actomyosin-dependent tissue flattening which erases the buckle within tens of seconds, in both mono-and multi-layered epithelia. Strikingly, we identify a well-defined limit to this response, so that stable folds form in the tissue when compressive strains exceed a 'buckling threshold' of ~35%. A combination of experiment and modelling shows that this behaviour is orchestrated by adaptation of the actomyosin cytoskeleton as it re-establishes tissue tension following compression. Thus, tissue pre-tension allows epithelia to both buffer against deformation and sets their ability to form and retain folds during morphogenesis.