Shen Jiayu, Goovaerts Quinten, Ajjugal Yogeeshwar, De Wijngaert Brent, Das Kalyan, Patel Smita S
Department of Biochemistry and Molecular Biology, Robert Wood Johnson Medical School, Rutgers University, Piscataway, NJ 08854, USA; Graduate School of Biomedical Sciences at the Robert Wood Johnson Medical School of Rutgers University, Piscataway, NJ 08854, USA.
Molecular Structural and Translational Virology, Department of Microbiology, Immunology and Transplantation, Rega Institute for Medical Research, KU Leuven, 3000 Leuven, Belgium.
Mol Cell. 2025 Jul 22. doi: 10.1016/j.molcel.2025.07.002.
Transcription of the human mitochondrial DNA is initiated by POLRMT and initiation factors mitochondrial transcription factor A (TFAM) and mitochondrial transcription factor B2 (TFB2M). We present cryo-electron microscopy (cryo-EM) structures of three transcription initiation intermediates (pre-catalytic IC3 [pre-IC3], slipped-IC3, and slipped pre-IC4) catalyzing RNA synthesis by normal and slippage pathways with fully resolved transcription bubbles and RNA transcripts starting from the +1 or -1 position. The structural and biochemical studies reveal mechanisms of promoter melting, start site selection, and slippage synthesis. Promoter melting begins at -4 with base-specific interactions of template -4 and -3 guanines with POLRMT and non-template -1 adenine with TFB2M. The NT-stabilizing loop (KLDPRSGGVIKPP) and Y209 of TFB2M and W1026 of POLRMT interact with the non-template strand to guide initiation from the +1 start site. The -1 position is not an alternative start site but supports slippage initiation by base-pairing with a slipped or rebound 2-nt RNA. Cryo-EM resolved additional apo and dimeric complexes whose populations may regulate transcription initiation.
人类线粒体DNA的转录由POLRMT以及起始因子线粒体转录因子A（TFAM）和线粒体转录因子B2（TFB2M）启动。我们展示了三种转录起始中间体（预催化IC3 [pre-IC3]、滑移IC3和滑移预IC4）的冷冻电镜（cryo-EM）结构，它们通过正常和滑移途径催化RNA合成，转录泡完全解析，RNA转录本从+1或-1位置开始。结构和生化研究揭示了启动子解链、起始位点选择和滑移合成的机制。启动子解链从-4开始，模板-4和-3鸟嘌呤与POLRMT以及非模板-1腺嘌呤与TFB2M之间存在碱基特异性相互作用。TFB2M的NT稳定环（KLDPRSGGVIKPP）和Y209以及POLRMT的W1026与非模板链相互作用，以引导从+1起始位点开始的起始过程。-1位置不是替代起始位点，但通过与滑移或回跳的2-nt RNA碱基配对来支持滑移起始。冷冻电镜解析了额外的无配体和二聚体复合物，其数量可能调节转录起始。
