Human mitochondrial RNA polymerase structures reveal transcription start-site and slippage mechanism

Human mitochondrial RNA polymerase (POLRMT) and protein factors TFAM and TFB2M assemble on mitochondrial DNA promoters to initiate promoter-specific transcription. We present cryo-EM structures of two initiation complexes, IC3 and slipped-IC3, with fully resolved transcription bubbles containing RNA transcripts starting from +1 and -1 positions, respectively. These structures reveal the mechanisms of promoter melting, start site selection, and slippage synthesis. Promoter melting begins at -4 with base-specific interactions of -4 and -3 template guanines with POLRMT and -1 non-template adenine with TFB2M, stabilizing the bubble and facilitating initiation from +1. Slippage occurs when a synthesized 2-mer RNA shifts to -1; the -1 position is not an alternative start-site. The conserved non-template sequence (-1)AAA(+2) is recognized by a non-template stabilizing loop (K₁₅₃LDPRSGGVIKPP₁₆₅) and Y209 from TFB2M and W1026 of POLRMT. The initiation complex on cryo-EM grids exist in equilibrium with apo and dimeric POLRMTs, whose relative concentrations may regulate transcription initiation.