Assembly of an active group II intron-maturase complex by protein dimerization.
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.
Rambo
Robert P
RP
Department of Molecular Biophysics and Biochemistry and Howard Hughes Medical Institute, Yale University, New Haven, Connecticut 06520, USA.
Doudna
Jennifer A
JA
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D004251:DNA Transposable Elements / Q000235:genetics; D019281:Dimerization; D007438:Introns / Q000502:physiology*; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D011487:Protein Conformation; D012329:RNA, Bacterial / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D012261:Ribonucleoproteins / Q000737:chemistry / Q000378:metabolism; D055672:Static Electricity
D001426:Bacterial Proteins; D004251:DNA Transposable Elements; C099786:LtrB protein, Lactococcus lactis; D046911:Macromolecular Substances; D012329:RNA, Bacterial; D012261:Ribonucleoproteins; C099785:LtrA protein, Lactococcus lactis; D012194:RNA-Directed DNA Polymerase