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Isolation of mRNAs Associated with Yeast Mitochondria to Study Mechanisms of Localized Translation
14:44

Isolation of mRNAs Associated with Yeast Mitochondria to Study Mechanisms of Localized Translation

Published on: March 14, 2014

Joint X-ray and NMR refinement of the yeast L30e-mRNA complex

Jeffrey A Chao1, James R Williamson

  • 1Department of Molecular Biology, The Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.

Structure (London, England : 1993)
|July 10, 2004
PubMed

Insights

This study reveals how Saccharomyces cerevisiae ribosomal protein L30e regulates gene expression by binding RNA. A refined structural analysis corrected previous errors, showing the RNA adopts a canonical K-turn fold.

Area of Science:

  • Molecular Biology
  • Structural Biology
  • Biochemistry

Background:

  • Ribosomal protein L30e regulates its own expression in Saccharomyces cerevisiae.
  • This regulation involves binding to a specific purine-rich RNA loop in pre-mRNA and mature mRNA.
  • Previous structural studies yielded conflicting RNA conformations.

Purpose of the Study:

  • To determine the accurate structural basis of the interaction between ribosomal protein L30e and its regulatory RNA element.
  • To resolve discrepancies between existing NMR and crystal structures.
  • To refine the RNA structure using a combination of experimental data.

Main Methods:

  • X-ray crystallography was used to solve the structure of an MBP-L30e fusion protein complexed with the regulatory RNA at 3.24 Å resolution.
  • Nuclear Magnetic Resonance (NMR) data was re-analyzed to identify and correct an erroneous imino proton resonance assignment.
  • Joint refinement of NMR and X-ray diffraction data was performed.

Main Results:

  • The crystal structure revealed an RNA conformation distinct from the previously determined NMR structure.
  • Re-analysis of NMR data identified a single misassigned imino proton resonance responsible for structural inaccuracies.
  • Joint refinement using both X-ray and NMR data improved stereochemistry and reduced crystallographic R factors.
  • The refined structure confirmed the RNA internal loop adopts a canonical K-turn fold.

Conclusions:

  • The accurate structure of the L30e-mRNA complex reveals a canonical K-turn fold for the RNA regulatory element.
  • This study corrects a significant error in previous structural assignments, providing a reliable model for L30e autoregulation.
  • The findings enhance our understanding of post-transcriptional gene regulation by ribosomal proteins.

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