Time-resolved small angle X-ray scattering

  • Brenowitz, Michael D. (PI)
  • Pollack, Lois (PI)
  • Steven, Chu (PI)
  • Altman, Russ Biagio (PI)
  • Doniach, Sebastian (PI)
  • Herschlag, Daniel (PI)

Project: Research project

Project Details

Description

RNA plays an important role in many biological processes, but its conformational dynamics are not yet understood on the most fundamental level: self-assembly or folding. The long term objective of this Program Project is the complete characterization of folding of a large RNA: the Tetrahymena ribozyme. This proposal presents aseries of studies designed to probe the global conformation of this ribozyme as it proceeds towards folding through an initial rapid collapse, and subsequent significant compaction steps. The use of a demonstrated combination of flow cells, designed for synchrotron small angle x-ray scattering, allows access to structural information on time scales ranging from sub-millisecond to minutes after the initiation of folding.
Previous work shows significant changes in the scattering signals throughout this regime, corresponding to large scale structural rearragements associated with compaction and folding.
Three specific aims are presented to achieve our long term goal. To realize the first, time resolved small angle x-ray scattering will be used to characterize the rapid collapse that occurs at the onset of folding. Rate constants and transient structural information will be obtained under different solution and sequence conditions to investigate the role of counterions in collapse and to test structural models of compact states. The second specific aim is directed at structural characterization of later intermediates including partially folded states, using small angle x-ray scattering. The reverse process of unfolding will also be studied. The final specific aim probes the global conformations along the preferred folding pathways that are populated in the presence of both monovalent and divalent cations. Solution conditions that more closely resemble physiological will be explored under this aim. Folding will be initiated from discrete sets of unfolded states to determine the characteristics of transients along separate pathways. The global structural information from these small angle scattering experiments will be integrated with detailed, local information acquired from other Project members to achieve the deepest possible understanding of the folding of this molecule.
StatusFinished
Effective start/end date1/1/013/31/20

ASJC

  • Computational Mathematics
  • Radiation
  • Molecular Biology
  • Biochemistry
  • Computer Science Applications
  • Physics and Astronomy(all)
  • Chemistry(all)
  • Spectroscopy

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