Structural insight of transcriptional terminator recognition by ProQ/FinO domain RNA chaperones

• Author / Creator

  Kim, Hyeong Jin

• The ProQ/FinO family is an emerging group of RNA binding proteins that are critical for sRNA-mediated gene regulation in proteobacteria. The ProQ/FinO domain of these proteins recognizes a transcriptional terminator, hairpin-tail structure, to regulate gene expression. The important role of the ProQ/FinO domain in RNA binding was well-studied, however, the molecular mechanism remained unclear due to a lack of structural information of ProQ/FinO proteins in complex with their target RNAs. To understand how the ProQ/FinO domain recognizes RNA in molecular detail, we chose to study RocC, a ProQ/FinO domain containing protein and its sRNA partner RocR.
  The structure of the ProQ/FinO domain of RocC in complex with the RocR transcriptional terminator was determined using X-ray crystallography at 3.2 Å resolution. Two conserved structural motifs within RocC were identified that were the major contact points for RocR. One was an N-cap motif which recognized the A-form structure of one strand at the base of the RocR hairpin. The other was a positively charged pocket containing a -turn--helix motif that recognized the last two 3’ nucleotides of the 3’ single-stranded tail.
  Structure-guided mutagenesis demonstrated essential residues for RocC:RocR binding in vitro and transformation repression in vivo. As the structure revealed, mutations in the N-cap motif and the 3’ binding pocket weakened binding and impaired biological function of RocC. Binding assays of RocC with a 10 nucleotide single-stranded RNA, which showed direct contact with RocC in the crystal structure, was tested using isothermal calorimetry (ITC). The dissociation constant (KD) decreased 19-fold compared to the intrinsic terminator, suggesting that A-form helical structure is required for tight binding to restrain conformation of 3’ side lower stem. The substitution of the 3’ hydroxyl group to a phosphate completely disrupted binding with RocC. This result corroborated the 3’ hydroxyl group recognition by RocC observed in crystal structure.
  An analysis of the conservation of the RocC RNA contact surfaces across the ProQ/FinO family suggests that the principles of RNA recognition from the RocC:RocR structure will hold for many members of this family.

• Subjects / Keywords

  • crystallography
  • RNA chaperone
  • RNA binding protein
  • ProQ
  • FinO

• Graduation date

  Fall 2022

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/r3-s0y5-kg59

• License

  This thesis is made available by the University of Alberta Library with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.