Cryo-EM structures reveal transcription initiation steps by yeast mitochondrial RNA polymerase

Understanding of mitochondrial DNA transcription initiation lags that of bacterial and nuclear DNA transcription. We have solved the structures of yeast mitochondrial RNA polymerase transcription pre-initiation complex (PIC) and initiation complex (IC) with fully resolved transcription bubbles. The structures explain promoter melting, template alignment, DNA scrunching, transition into elongation, and abortive synthesis. Partially melted DNA in PIC is caused by interactions of MTF1 with four flipped non-template bases. In IC, the bubble expands, a largescale movement aligns the template with RNA/NTP in the active site, and the downstream DNA is repositioned. The IC-state captures the scrunched DNA as an NT-loop that interacts with the centrally positioned MTF1 C-tail. Coexisting scrunched and unscrunched states and imminent steric clashes of NT-loop and RNA:DNA with C-tail explain abortive synthesis and transition into elongation. The IC-state with UTPαS poised for catalytic incorporation provides a platform to assess the toxicity of antiviral nucleosides/nucleotides like remdesivir.