Lisica Ana, Engel Christoph, Jahnel Marcus, Roldán Édgar, Galburt Eric A, Cramer Patrick, Grill Stephan W
Biotechnology Center, Technical University Dresden, 01307 Dresden, Germany; Max Planck Institute of Molecular Cell Biology and Genetics, 01307 Dresden, Germany;
Department of Molecular Biology, Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany;
Proc Natl Acad Sci U S A. 2016 Mar 15;113(11):2946-51. doi: 10.1073/pnas.1517011113. Epub 2016 Feb 29.
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.
在DNA转录过程中，RNA聚合酶常常会进入无活性的回溯状态。从回溯状态恢复可通过一维扩散或切割回溯的RNA来实现，但聚合酶如何做出这种选择尚不清楚。在这里，我们利用单分子光镊实验和随机理论表明，回溯恢复机制的选择是由一维扩散和RNA切割之间的动力学竞争决定的。值得注意的是，RNA聚合酶I（Pol I）和Pol II通过一维扩散从浅回溯中恢复，利用RNA切割从中等深度的回溯中恢复，并且无法从广泛的回溯中恢复。此外，Pol I和Pol II使用不同的机制来避免不可恢复的回溯。Pol I受其亚基A12.2的保护，该亚基降低了一维扩散的速率，并使转录本切割可达20个核苷酸。相比之下，Pol II通过与切割刺激因子TFIIS结合而得到完全保护，TFIIS能够通过RNA切割从任何深度快速恢复。综上所述，我们确定了Pol I和Pol II不同的回溯恢复策略，为这些关键酶的细胞功能进化提供了线索。
