Abstract

Group II intron-encoded proteins promote both splicing and mobility of the intron RNA through formation of a specific RNA-protein complex. The Lactococcus lactis L1.LtrB intron encodes a maturase, LtrA, with reverse transcriptase homology and specific binding affinity for two domains of the intron RNA. The catalytically active ribonucleoprotein (RNP) has splicing, endonuclease, and reverse transcriptase activity, enabling efficient insertion of the intron sequence by a retro-homing mechanism. To determine the composition and assembly mechanism of the RNP complex, purified LtrA protein was analyzed for its ability to recognize a series of intron-derived RNAs. Equilibrium dissociation measurements show that LtrA recognizes two intronic domains, DI and DIV. However, distinct electrostatic requirements for binding imply different modes of molecular recognition in each case. Stoichiometric binding experiments show that the functional RNP complex consists of a dimeric protein species bound to a single intron RNA. Significant differences between the measured equilibrium dissociation constants and kinetically derived values suggest that conformational changes accompany assembly of the intron-maturase complex, and results of limited proteolysis and fluorescence spectroscopy experiments suggest that significant RNA-dependent structural changes within the maturase occur upon association with the intron. These results support a mutually induced fit model in which RNA-dependent conformational changes within LtrA enable stable association of the protein dimer with two independent intron domains to form a functional RNP.

Talk to us

Join us for a 30 min session where you can share your feedback and ask us any queries you have

Schedule a call

Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.