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

Brent De Wijngaert; Shemaila Sultana; Chhaya Dharia; Hans Vanbuel; Jiayu Shen; Daniel vasilchuk; Sergio E Martinez; Eaazhisai Kandiah; Smita S Patel; Kalyan Das

The PIC structure has not been observed previously; thus, it provides new insights into the mechanism of promoter melting. The y-mtPIC structure suggests that RPO41 and MTF1 initiate the promoter melting by creating a 4-nt transcription bubble from -4 to -1. We provided a -4 to +2 pre-melted promoter; however, the +1 and +2 nucleotides assume a duplex-like DNA conformation in PIC albeit lacking canonical base-pairing. DNA melting is driven by sequence-specific interactions of the NT strand with the NT-groove that lies at the interface of N-and C-terminal domains of MTF1 (residues 103-105, 144-148, and 190-192) . The -4 to -2 AAG bases in the NT strand are flipped towards NT-groove (Fig. 1G ). The -2 guanine base is sandwiched between the aromatic side chains of Y103 and W105, and all N and O atoms of the base, except N7, are engaged in complementary hydrogen bond interactions (Fig. 1H) ; mutation of -2 guanine severely impair promoter melting (9). The -1 NT base stacks with Y103 with no base-specific interaction. The -3 and -4 AA bases are The structure reveals that the aromatic sidechain of mutated E144F or C192F interferes with the binding of NT -3 and -4 bases and R178A+K179A would reduce DNA-backbone interactions.