Activating the branch-forming splicing pathway by reengineering the ribozyme component of a natural group II intron

RNA
Dario MonachelloMaria Costa

Abstract

When assayed in vitro, group IIC self-splicing introns, which target bacterial Rho-independent transcription terminators, generally fail to yield branched products during splicing despite their possessing a seemingly normal branchpoint. Starting with intron O.i.I1 from Oceanobacillus iheyensis, whose crystallographically determined structure lacks branchpoint-containing domain VI, we attempted to determine what makes this intron unfit for in vitro branch formation. A major factor was found to be the length of the helix at the base of domain VI: 4 base pairs (bp) are required for efficient branching, even though a majority of group IIC introns have a 3-bp helix. Equally important for lariat formation is the removal of interactions between ribozyme domains II and VI, which are specific to the second step of splicing. Conversely, mismatching of domain VI and its proposed first-step receptor in subdomain IC1 was found to be detrimental; these data suggest that the intron-encoded protein may promote branch formation partly by modulating the equilibrium between conformations specific to the first and second steps of splicing. As a practical application, we show that by making just two changes to the O.i.I1 ribozyme, it is possible to...Continue Reading

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Citations

May 13, 2017·The International Journal of Biochemistry & Cell Biology·Matthew NguLinda Bonen
Dec 10, 2016·Science·Maria CostaFrançois Michel
Aug 21, 2021·Biochemical Society Transactions·Dulce Alonso, Alfonso Mondragón

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