Important 2'-hydroxyl groups within the core of a group I intron.
The catalytic activity of a group I intron is dependent on a core structure, much of which is not exposed to solvent. In order to study the structure of the core, an efficient bimolecular reaction has been developed: the 5'-component is a molecule of about 300 bases which contains the 5'-splice-site and terminates in the loop established by P8, and the 3'-component is a 24 base long oligoribonucleotide which includes the 3'-regions of the P8 and P7 helices with their joining region, J8/7. J8/7 is thought to play several roles including binding the helix containing the 5'-splice-site. P7 forms a major portion of the guanosine binding site required for splicing. We have modified the bimolecular system to make it amenable to kinetic analysis and have used it to study the role of the ribose sugars in the oligomer. Multiple deoxyribonucleotide substitution in the J8/7 region completely blocked 5'-splice-site cleavage even though the Kd was only reduced about 5-fold. This supports the idea that the ribose phosphate backbone in J8/7 plays a key role in catalysis. Individual substitutions at G303 and A306 reduced the rate of catalysis 5-10-fold. The G303 substitution blocked GTP-independent hydrolysis of the 5'-splice-site. The region spanning the junction of P8 and J8/7 was also highly sensitive to multiple deoxyribonucleotide substitution; however, only in the case of C298 did an individual substitution have any effect on cleavage. Deoxyribonucleotide substitution in the 3'-section of P7 was less severe, although kcat/Km in low GTP was down 70-fold.
Caprara
M G
MG
Department of Biology, Temple University, Philadelphia, Pennsylvania 19122.
Waring
R B
RB
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D006160:Guanosine Triphosphate / Q000378:metabolism; D006900:Hydroxylation; D007438:Introns; D007700:Kinetics; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D012322:RNA Precursors / Q000737:chemistry; D012326:RNA Splicing; D016337:RNA, Catalytic / Q000737:chemistry* / Q000378:metabolism; D016053:RNA, Protozoan / Q000737:chemistry* / Q000378:metabolism; D016808:Tetrahymena thermophila / Q000235:genetics*
D012322:RNA Precursors; D016337:RNA, Catalytic; D016053:RNA, Protozoan; D006160:Guanosine Triphosphate