| Several group II introns have been shown to self-splice in vitro , but genetic evidence suggests that most require trans-acting factors to splice efficiently in vivo. Characterization of these factors will be important for understanding the roles of accessory factors in modulating the functions of catalytic RNAs. I have identified a nuclear gene in maize, crs2, that is required for the splicing of many group II introns; in chloroplasts. Analysis of crs2 mutants and mutants lacking plastid ribosomes showed that there are two classes of chloroplast introns in maize: those whose splicing is dependent upon crs2 function and those whose splicing requires plastid ribosomes. Group II introns have been divided into two subgroups, subgroup IIA and subgroup IIB, based on distinct structural features. All of the introns that exhibit a strict dependence on crs2 function fall into group IIB while those whose splicing correlates with the presence of plastid ribosomes fall into group IIA. Thus, there may be structural elements common to group HA introns that are recognized by a chloroplast-encoded factor and different structural elements in group IIB introns that are recognized by one or more factors whose activity requires crs2 function.; To understand the biochemical role of crs2, I cloned the crs2 gene by transposon tagging. The crs2 cDNA sequence indicates that CRS2 is homologous to peptidyl-tRNA hydrolases (PTH). These are essential proteins in bacteria that cleave an ester linkage between the tRNA and nascent peptide on peptidyl-tRNAs that accumulate as abortive translation products. CRS2 may not function as a PTH since it cannot complement a temperature-sensitive pth mutant in E. coli. Fractionation experiments indicate that CRS2 is a stromal protein that is in a macro-molecular complex of 575–800 Microccocal nuclease treatment shifts CRS2 into a complex of 55–81 kD, which is larger than the 23 kD CRS2 monomer. These results indicate that CRS2 is in a complex with RNA, and that this complex likely contains multiple polypeptides. This dissertation contains co-authored and previously published material. |
