RNA and Protein Catalysis in Group II Intron Splicing and Mobility Reactions Using Purified Components

• Published in: Biochemistry (Easton) 1999-07, Vol.38 (28), p.9069-9083
• Main Authors: Saldanha, Roland, Chen, Bing, Wank, Herbert, Matsuura, Manabu, Edwards, Judy, Lambowitz, Alan M
• Format: Article
• Language: English
• Subjects: Bacterial Proteins - genetics; Bacterial Proteins - isolation & purification; Bacterial Proteins - metabolism; Catalysis; Deoxyribonucleases, Type II Site-Specific - genetics; Deoxyribonucleases, Type II Site-Specific - metabolism; DNA Transposable Elements; Enzyme Stability - genetics; Escherichia coli; Escherichia coli - genetics; Hydrogen-Ion Concentration; Introns; Lactococcus lactis; Nucleic Acid Conformation; Recombinant Proteins - biosynthesis; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Ribonucleoproteins - genetics; Ribonucleoproteins - metabolism; RNA Splicing; RNA, Bacterial - chemistry; RNA, Bacterial - genetics; RNA, Bacterial - metabolism; RNA-Binding Proteins - metabolism; RNA-Directed DNA Polymerase - genetics; RNA-Directed DNA Polymerase - metabolism; Sodium Chloride - chemistry; Substrate Specificity - genetics
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi982799l

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 °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.