RNA and protein catalysis in group II intron splicing and mobility reactions using purified components.
Group II introns encode proteins with reverse transcriptase activity. These proteins also promote RNA splicing (maturase activity) and then, with the excised intron, form a site-specific DNA endonuclease that promotes intron mobility by reverse splicing into DNA followed by target DNA-primed reverse transcription. Here, we used an Escherichia coli expression system for the Lactococcus lactis group II intron Ll.LtrB to show that the intron-encoded protein (LtrA) alone is sufficient for maturase activity, and that RNP particles containing only the LtrA protein and excised intron RNA have site-specific DNA endonuclease and target DNA-primed reverse transcriptase activity. Detailed analysis of the splicing reaction indicates that LtrA is an intron-specific splicing factor that binds to unspliced precursor RNA with a K(d) of </=0.12 pM at 30 degrees C. This binding occurs in a rapid bimolecular reaction, which is followed by a slower step, presumably an RNA conformational change, required for splicing to occur. Our results constitute the first biochemical analysis of protein-dependent splicing of a group II intron and demonstrate that a single intron-encoded protein can interact with the intron RNA to carry out a coordinated series of reactions leading to splicing and mobility.
Saldanha
R
R
Institute for Cellular and Molecular Biology, Departments of Chemistry and Biochemistry, and Section of Molecular Genetics and Microbiology, School of Biological Sciences, University of Texas at Austin, Austin, Texas 78712, USA.
Chen
B
B
Wank
H
H
Matsuura
M
M
Edwards
J
J
Lambowitz
A M
AM
D001426:Bacterial Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D002384:Catalysis; D004251:DNA Transposable Elements; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics / Q000378:metabolism; D004795:Enzyme Stability / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007438:Introns; D013294:Lactococcus lactis; D009690:Nucleic Acid Conformation; D012326:RNA Splicing; D012329:RNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D012261:Ribonucleoproteins / Q000235:genetics / Q000378:metabolism; D012965:Sodium Chloride / Q000737:chemistry; D013379:Substrate Specificity / Q000235:genetics
D001426:Bacterial Proteins; D004251:DNA Transposable Elements; C099786:LtrB protein, Lactococcus lactis; D012329:RNA, Bacterial; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D012261:Ribonucleoproteins; D012965:Sodium Chloride; C099785:LtrA protein, Lactococcus lactis; D012194:RNA-Directed DNA Polymerase; D015252:Deoxyribonucleases, Type II Site-Specific