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Yeast rRNA Expansion Segments: Folding and Function.

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The largest ribosomal RNA expansion segment (ES7) in yeast ribosomes binds many proteins crucial for cellular functions, including those involved in stress response and gene expression. This suggests ES7 plays a key role in ribosome functionality.

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SHAPEaminoacyl tRNA synthetasesrRNA expansion segmentsrRNA‐protein interactionthermal unfolding

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Area of Science:

  • Molecular Biology
  • Cell Biology
  • Biochemistry

Background:

  • Eukaryotic ribosomal RNA (rRNA) contains expansion segments (ESs) absent in prokaryotes, contributing to larger ribosome size.
  • These ESs, particularly eukaryotic large ribosomal subunit's ES7, are located on the ribosome surface and are highly variable.
  • We hypothesize that these larger eukaryotic rRNAs, specifically ES7, enhance ribosome functionality.

Purpose of the Study:

  • To characterize the protein binding partners of Saccharomyces cerevisiae expansion segment 7 (ES7).
  • To investigate the potential role of ES7 as a hub for non-ribosomal proteins essential for eukaryotic ribosomal function.
  • To explore ES7's potential analogy to the multi-synthetase complex in higher organisms.

Main Methods:

  • In vitro RNA-protein pull-down assays using ES7 as bait.
  • Affinity confirmation using in vitro binding assays for specific synthetases.
  • Thermal denaturation and footprinting experiments to assess ES7 stability and structure.

Main Results:

  • ES7 acts as a binding hub for diverse non-ribosomal proteins involved in abiotic stimulus response, ribosomal biogenesis, protein transport, and transcription elongation.
  • Seven aminoacyl-tRNA synthetases (Ala-, Arg-, Asp-, Asn-, Leu-, Lys-, TyrRS) were identified as ES7-associated proteins.
  • Binding affinities for AspRS, TyrRS, and LysRS were confirmed; isolated ES7 demonstrated structural stability.

Conclusions:

  • S. cerevisiae ES7 associates with proteins critical for various cellular processes, suggesting a broader functional role beyond rRNA structure.
  • ES7 may function analogously to the multi-synthetase complex, integrating cellular functions with ribosome activity.
  • ES7 is structurally stable and plays a significant role in the overall functionality of eukaryotic ribosomes.