Related Experiment Videos
Group II intron reverse transcriptase in yeast mitochondria. Stabilization and regulation of reverse transcriptase
S Zimmerly1, J V Moran, P S Perlman
1Departments of Molecular Genetics and Biochemistry, The Ohio State University, Columbus, OH, 43210, USA.
Journal of Molecular Biology
|June 5, 1999
Summary
Group II introns utilize reverse transcriptases for splicing and mobility. This study identifies a 62 kDa protein (p62) in yeast mitochondria, showing its RNA binding stabilizes and regulates this crucial reverse transcriptase activity.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Group II introns are mobile genetic elements that utilize self-encoded reverse transcriptases for RNA splicing and DNA integration.
- These reverse transcriptases are essential for intron mobility and play a role in the splicing of precursor RNA.
Purpose of the Study:
- To characterize the reverse transcriptase activity associated with the yeast mitochondrial group II intron aI2.
- To investigate the role of the intron-encoded protein p62 in the binding, stability, and regulation of this reverse transcriptase activity.
Main Methods:
- Immunoblotting and UV cross-linking experiments to identify and characterize the reverse transcriptase-associated protein.
- RNase digestion to assess the dependence of reverse transcriptase activity on bound RNAs.
- Non-denaturing gel electrophoresis and activity assays to determine the association of reverse transcriptase with different RNA species.
- In vitro assays using DNA oligonucleotides and target DNA sites to study reverse transcription templates.
Main Results:
- The reverse transcriptase activity of the yeast mtDNA group II intron aI2 is encoded by a 62 kDa protein (p62).
- p62 is tightly bound to endogenous RNAs within mitochondrial ribonucleoprotein particles, and this binding is essential for maintaining reverse transcriptase activity.
- The reverse transcriptase is predominantly associated with excised intron RNA, with a smaller fraction bound to precursor RNA, and its activity is regulated to prevent endogenous RNA transcription without a primer.
- Mutations can bypass this regulation, leading to reverse transcription of endogenous RNAs, and the reverse-spliced intron RNA serves as the major template for target DNA-primed reverse transcription.
Conclusions:
- Binding to intron-containing RNAs is critical for stabilizing the p62 reverse transcriptase and regulating its enzymatic activity.
- The interaction between p62 and its cognate RNAs ensures proper function in both RNA splicing and intron mobility.
- Understanding this regulation provides insights into the mechanisms of group II intron propagation and reverse transcriptase function.