X-ray crystal structures explain DNA-hairpin promoter binding and de novo transcription initiation for the N4 virion RNA polymerase.

摘要

N4 vRNAP is a virion-encapsidated RNA polymerase (vRNAP) from the N4 bacteriophage that is injected into host E. coli K12 cells to transcribe N4 early genes. Its central domain (mini-vRNAP) contains all RNAP functions of the full-length vRNAP, which recognizes a five- to seven-base pair stem and three-nucleotide loop hairpin DNA promoter. I have determined X-ray crystal structures of promoter bound and transcription initiation complexes of N4 mini-vRNAP at 2.0 and 1.8 A, respectively. Structural motifs in N4 mini-vRNAP participating in promoter recognition are similar to those found in the T7 RNA polymerase, although T7 recognizes a double-stranded DNA promoter. The binary complex structure revealed that the plug and motif B loop, which block the access of template DNA to the active site in the apo-form mini-vRNAP, undergo a large-scale conformational change upon promoter binding, explaining the restricted promoter specificity that is critical for N4 phage early transcription.;While much is known about RNA polymerization with a pre-existing primer (called transcription elongation), the structural study presented in this thesis elucidates how the RNA polymerase synthesizes RNA without a primer (de novo transcription initiation). Here I report the first transcription initiation complex for a DNA-dependent RNA polymerase - N4 mini-vRNAP- that has both initiating nucleotides as well as both catalytic metals in the active site. This structure provides for a detailed analysis of the conformations and interactions of both the substrates and protein side chains. I report pre-catalytic de novo transcription initiation complexes, with and without catalytic metal A, that reveal that the gamma phosphate group of the +1 nucleotide is involved in metal binding and positions proximal to its own 3'-OH group, suggesting a possible role in catalysis. X-ray crystallographic analysis of a K437A N4 mini-vRNAP mutant transcription initiation complex revealed that K437 plays a role in +1 triphosphate stabilization. Lastly, I have solved the structure of a pre-translocated 2-mer product and pyrophosphate transcription initiation complex that completes a structural description of the beginning and end events of the first phosphodiester bond formation.