Poster abstracts
Poster number 111 submitted by Jasmine Harper
Mechanistic Coupling of cDNA Synthesis and Transesterification Drives Group II Intron Retromobility
Jasmine A. Harper (West Virginia University, Department of Biochemistry and Molecular Medicine), Sarah A. Starcovic (West Virginia University, Department of Biochemistry and Molecular Medicine), Aaron R. Robart (West Virginia University, Department of Biochemistry and Molecular Medicine)
Abstract:
Group II introns are catalytic RNAs (ribozymes) known to self-splice out of precursor RNA and to mobilize into a DNA target. To facilitate their activities, these ribozymes undergo major conformational changes in their tertiary structure. Additionally, many group II introns encode and rely on an intron-encoded protein (IEP). The IEP acts as a maturase that facilitates proper intron folding for splicing and possesses reverse transcriptase activity essential for mobility. Together, the IEP and intron RNA form a ribonucleoprotein (RNP) complex, which serves as the entry molecule for intron insertion into a DNA target via retromobility. Retromobility proceeds by two transesterification steps followed by IEP-catalyzed cDNA synthesis, in which the IEP uses the intron as a template to complete insertion. We observed a reaction bottleneck between the two transesterification steps that prevents sufficient completion of the second step. Using the group II intron Ta.it.I1, we observed that the second transesterification step is dependent on IEP-catalyzed cDNA synthesis. We determined that the intron-exon junction serves as the threshold that cDNA synthesis must cross to facilitate the second step. Therefore, rather than being distinct sequential steps, the second transesterification step and cDNA synthesis are coupled reactions. Because these steps require major conformational changes in the group II intron structure, we employed cryogenic electron microscopy to resolve two structures of the RNP stalled at pre-second step and post-second step states. This work redefines the fundamental mechanism of group II intron retromobility and provides a structural framework for understanding RNP coordination during target DNA insertion.
Keywords: group II introns, retromobility, cryo-EM