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Site-Directed Mutagenesis for In Vitro and In Vivo Experiments Exemplified with RNA Interactions in Escherichia Coli
Published on: February 5, 2019
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Modulation of conformational changes in helix 69 mutants by pseudouridine modifications
Jun Jiang1, Daya Nidhi Kharel1, Christine S Chow1
1Department of Chemistry, Wayne State University, Detroit, MI 48202, United States.
Biophysical Chemistry
|March 25, 2015
Summary
Pseudouridine modifications and sequence changes impact ribosomal helix 69 structure and function during translation. Pseudouridine stabilizes helix 69 at low pH, influencing ribosome activity.
Area of Science:
- Molecular Biology
- Biophysics
- Structural Biology
Background:
- Helix 69 (H69) of 23S rRNA is crucial for translation, located at the ribosomal subunit interface and mRNA tunnel.
- Three pseudouridine modifications influence H69 structure and conformational dynamics, affecting ribosome activity.
Purpose of the Study:
- To investigate the combined effects of pseudouridine and sequence variations on H69 biophysical properties.
- To analyze how mutations (A1912G, U1917C, A1919G) and pseudouridine affect H69 structure and stability.
Main Methods:
- Nuclear Magnetic Resonance (NMR) spectroscopy.
- Circular Dichroism (CD) spectroscopy.
- Analysis of wild-type and mutant H69 constructs.
Main Results:
- pH-dependent structural changes in H69 are modulated by pseudouridine and loop sequence.
- Mutations had minimal impact on H69 global stability.
- Pseudouridine significantly stabilizes H69 under acidic (low pH) conditions.
Conclusions:
- Pseudouridine and specific loop sequences cooperatively modulate H69 structure and pH-dependent behavior.
- While mutations alone do not compromise stability, altered conformational dynamics due to pseudouridine may impact H69 function in translation.
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