Yeast mtDNA transcription initiation in single nucleotide addition steps

Transcription initiation catalyzed by the RNA polymerase is a multistep process involving promoter binding, transcription bubble formation, abortive RNA synthesis, and transition into elongation following promoter escape. We report cryo-EM structures of yeast mitochondrial RNA polymerase initiation complexes (ICs) with transcription factor MTF1 catalyzing RNA synthesis from de novo initiation to 6-mer synthesis at single-nucleotide steps on fully-resolved transcription bubbles. The growing RNA:DNA hybrid is accommodated by continuous scrunching of the template strand while the non-template and MTF1 C-tail in the polymerase cleft are structurally reorganized. Each nucleotide addition accumulates stress energy, which drives abortive RNA synthesis during early transcription initiation steps and promoter release later. The non-template scrunches as loops in IC2/IC3, and unscrunching assists abortive synthesis of 2-/3-mer RNAs. Subsequently, in IC5 and IC6, the non-template strand assumes a stable structure by stacking its bases into a spiral staircase-like structure that supports processive synthesis. In IC6, the template scrunches to the maximum and places the -1 nucleotide in a pocket near the thumb domain. Subsequently, the -1 nucleotide acts as a pivot point for promoter escape ushering the IC into the elongation phase. The structural snapshots visualize the interplay between abortive and productive synthesis regulating transcription initiation.