Hydrophobic unnatural base pair promotes trigger loop closure and catalysis in cellular RNA polymerase independent of hydrogen bonding

• 成果类型: Article
• 署名作者: Li, Qingrong; Hou, Peini; Kimoto, Michiko; Oh, Juntaek; Liu, Yan; Tan, Hui Pen; Chong, Jenny; Hirao, Ichiro; Wang, Dong
• 署名单位: University of California System; University of California San Diego; Kyung Hee University; Kyung Hee University; Kyung Hee University; United States Department of Energy (DOE); Stanford University; SLAC National Accelerator Laboratory; University of California System; University of California San Diego; University of California System; University of California San Diego
• 刊物名称: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• ISSN/ISSBN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.2607774123
• 发表日期: 2026-08-18
• 页码: e2607774123
• 关键词: transcription; unnatural base pair; trigger loop; hydrogen bond; synthetic biology; ALPHABET EXPANSION TRANSCRIPTION; SITE-SPECIFIC INCORPORATION; cryo-em; SYSTEM; molecules; dna; analogs

摘要

Expanded genetic alphabets based on synthetic nucleotides offer a powerful strategy to increase the chemical diversity and functional potential of nucleic acids. Hydrophobic unnatural base pairs (UBPs) represent a unique class of UBPs that operate without canonical hydrogen bonding and shapes, which are distinct from natural base pairs. However, the molecular mechanisms governing hydrophobic UBP recognition and incorporation by RNA polymerases remain poorly understood, because no structures of catalytically competent state have been captured. Here, we investigate the enzyme kinetics and structural basis of transcription recognition of hydrophobic 7-(2-thienyl)-imidazo[4,5-b]pyridine (Ds):pyrrole 2-carbaldehyde (Pa) base pair by the Escherichia coli RNA polymerase (EcRNAP). Enzyme kinetics reveal a pronounced strand-dependent asymmetry, with DsTP incorporated substantially more efficiently than PaTP. To elucidate the structural origin of this asymmetry, we determined two cryo-electron microscopy structures of E. coli RNAP elongation complexes containing dPa:DsTP and dDs:PaTP at resolutions of 3.20 & Aring; and 3.24 & Aring;, respectively. The dPa:DsTP structure captures a pre-catalytic state of hydrophobic UBP transcription in which DsTP forms an edge-to-edge base pair with templating dPa, and the active site adopts a native-like, catalytically competent configuration with fully folded trigger loop. In contrast, the dDs:PaTP structure reveals a pre-insertion intermediate with incomplete substrate loading. Structural modeling indicates that PaTP must undergo an additional insertion step to achieve productive catalysis. Together, these findings define the molecular basis of asymmetric Ds:Pa UBP transcription recognition by a multisubunit RNAP and provide structural principles for designing next-generation UBPs compatible with cellular transcription.