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Kinetic analysis of ribozyme-substrate complex formation in yeast
Ramesh S Yadava1, Elisabeth M Mahen, Martha J Fedor
1Department of Molecular Biology and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA 92037, USA.
RNA (New York, N.Y.)
|April 22, 2004
Summary
RNA complex assembly is crucial for many cellular processes but poorly understood. This study reveals that efficient intracellular RNA complex formation requires greater complementarity in vivo than in vitro, especially with high divalent cations.
Area of Science:
- Molecular Biology
- Biochemistry
- RNA Biology
Background:
- RNA-mediated reactions necessitate RNA complex assembly, yet pathways are unclear.
- Assessing RNA assembly in vivo is challenging due to complex interactions and experimental limitations.
Purpose of the Study:
- To investigate intracellular RNA complex formation and cleavage kinetics using chimeric U3 small nucleolar RNAs (snoRNAs) in yeast.
- To compare in vitro and in vivo RNA complex assembly efficiency under varying ionic conditions.
Main Methods:
- Expressed ribozyme and target sequences as separate chimeric U3 snoRNAs in yeast.
- Measured intracellular cleavage rates and compared them with in vitro kinetic parameters.
- Analyzed the effect of helix length and divalent cation concentration on RNA complex formation.
Main Results:
- Intracellular cleavage rates correlated with ribozyme-substrate complex formation via helix annealing.
- Efficient RNA complex formation in vivo required longer complementary regions than in vitro, particularly with high MgCl(2).
- Intracellular cleavage rates were 15- to 30-fold lower than in vitro, suggesting substrate binding is rate-limiting and complex formation is diffusion-limited in yeast nuclei.
Conclusions:
- Substrate binding is the rate-determining step for intracellular RNA cleavage.
- RNA complex formation in yeast nuclei appears to be diffusion-limited, consistent with U3 small nucleolar ribonucleoproteins (snoRNPs) diffusion coefficients.