The proposed research involves investigation of how group B intron protein maturases promote RNA splicing, using the Lactococcus lactis group II intron as a model. Group B introns are mobile genetic elements that splice via intron-encoded reverse transcriptases that also function as maturases. Recent development of an E. coli-based expression system for the L. Lactis group II intron, Ll.LtrB, and the protein maturase, denoted LtrA, has enabled the study of the splicing and mobility reactions of the Lactococcal intron in vivo, and in vitro using purified components. This research will use this expression system in combination with UV-crosslinking techniques and protein mutagenesis to investigate how the maturase folds the intron RNA into a catalytic structure to promote splicing, and to determine the specific regions of the RNA and protein that interact.
Specific aims are: (1) To investigate how the L. lactis maturase LtrA induces the conformational changes that are necessary for catalytic activity in the Ll.LtrB intron RNA, using UV- crosslinking experiments: . (2) To identify the regions that interact between Ll.LtrB and LtrA, using site-specific photoreagent placement and UV-crosslinking. (3) To investigate the function of protein domains in LtrA by analysis of LtrA mutant proteins.
| Noah, James W; Park, Soyeun; Whitt, Jacob T et al. (2006) Atomic force microscopy reveals DNA bending during group II intron ribonucleoprotein particle integration into double-stranded DNA. Biochemistry 45:12424-35 |
