Characterization of HelD, a Novel RNA polymerase Binding Protein in Bacillus subtilis

Open Access
Author:
Nguyen, Uyen Phong
Area of Honors:
Biochemistry and Molecular Biology
Degree:
Bachelor of Science
Document Type:
Thesis
Thesis Supervisors:
  • Katsuhiko Murakami, Thesis Supervisor
  • Ming Tien, Honors Advisor
Keywords:
  • biochemistry
  • x-ray crystallography
  • RNA polymerase
  • bacterial transcription
  • cryo EM
  • structural biology
Abstract:
RNA polymerase (RNAP) is an essential enzyme that is responsible for transcription of genes in all organisms. A protein called HelD (YvgS) has been recently identified as a transcription factor of RNAP in Gram-positive Bacillus subtilis, which enhances transcription by increasing RNAP recycling. Based on sequence homology, HelD belongs to the Superfamily 1 (SF1) DNA helicase. However, the mechanism of HelD-dependent transcription activation is limited due to lack of the structures of HelD and also the RNAP and HelD complex. The goal of this project is to obtain a high-resolution three-dimensional structure of HelD in complex with B. subtilis RNAP to elucidate the structural basis of RNAP recycling by the DNA helicase HelD. Two techniques have been used toward the structure determinations in this project: one is X-ray crystallography to determine the structure of HelD, and single-particle cryo-electron microscopy (cryo-EM) to determine the structure of HelD-RNAP complex. Truncations of HelD were constructed for protein expression to enhance protein crystallibility. DNA binding and unwinding function were assayed with HelD using electrophoretic mobility shift assay to ensure functional HelD was purified for downstream structural determination experiments. Overall, this project provides a framework for purification protocol of HelD. In future experiments, structure determination of HelD and HelD- RNAP complex would enhance our knowledge of bacterial transcription.