Mechanistic investigations of nucleotidyl-transfer reaction and de novo initiation in Rna polymerases by X-ray crystallography

Open Access
Basu, Ritwika
Graduate Program:
Biochemistry, Microbiology, and Molecular Biology
Doctor of Philosophy
Document Type:
Date of Defense:
December 12, 2013
Committee Members:
  • Katsuhiko Murakami, Dissertation Advisor
  • Katsuhiko Murakami, Committee Chair
  • Craig Eugene Cameron, Committee Member
  • Paul Lee Babitzke, Committee Member
  • Andrey S Krasilnikov, Committee Member
  • Scott A Showalter, Committee Member
  • soak-trigger freeze X-ray crystallography
  • nucleotidyl-transfer reaction
  • RNA polymerase
  • N4 bacteriophage
  • Thermus thermophilus
Nucleotidyl-transfer reaction is the fundamental reaction catalyzed by nucleic acid polymerases to synthesize RNA or DNA. Nucleotidyl transferases, including DNA and RNA polymerases (DNAP and RNAP) utilize the two-metal catalysis mechanism. However the temporal order of events in the reaction mechanism is not fully understood. Studies so far are based on predictions from biochemical studies and X-ray structural snapshots obtained with the use of non-reactive substrate analogues. This thesis presents a novel time-resolved X-ray crystallography approach to monitor the enzyme motions through high-resolution crystal structures. A soak-trigger freeze X-ray crystallography technique provides an atomic resolution movie of the active site events during the natural course of nucleotidyl-transfer reaction catalyzed by bacteriophage N4 mini-vRNAP. The structures are the first direct evidence of the temporal order of substrate assembly and metal co-factor binding at the active site of single-unit polymerases. The work reveals that the nucleotide substrates bind the active site along with MeB (nucleotide binding metal) and the catalytic metal (MeA) coordination is only temporary, established just before and dissociated immediately after phosphodiester bond formation. To expand the mechanistic studies to cellular multisubunit RNAPs, X-ray crystal structures of transcription initiation complexes from bacteria Thermus thermophilus, have been presented. Bacterial RNAP binds to initiation factor σ, forming the holoenzyme, which is capable of recognizing and binding to promoter DNA, leading to a transcription-ready open complex formation. The holoenzyme initiates RNA synthesis de novo, the only step during transcription where RNAP accepts two nucleotide substrates and performs primer-independent phosphodiester bond formation. The molecular mechanism of preorganization of the RNAP holoenzyme-promoter DNA complex in bacteria has been well studied, but there is no direct evidence for the mechanism of de novo initiation. I solved a structure of a de novo initiation complex that provides the first structural basis for the stable binding of the initiation nucleotide. The structure shows base stacking interactions between the DNA template at -1 position and the incoming nucleotide at +1, which explains the sequence preference of purine-pyrimidine for the -1 and +1 template positions of many RNAP promoters. Further, I solved the structure of a holoenzyme transcription complex containing a 6-mer transcript that was synthesized in crystallo. The structure demonstrates an early step of σ ejection from the RNAP core enzyme when transitioning to a stable transcription elongation complex. The structure shows that the RNA 5′-triphosphate end reaches the σ region of 3.2 causing it to be disordered, suggesting that 6-mer RNA transcription is a first step in this transition.