Structure and function studies on nrdB group I intron from bacteriophage T4

Sammanfattning: The nrdB gene of bacteriophage T4 contains a group I intron which is autocatalytic in water solutions containing salts. The group I intron self-splicing reaction consists of two consecutive transesterfication steps. The first step is the attack of an external guanosine on phosphor at the 5'-splice site. The external guanosine becomes covalently attached via its 3'-hydroxyl to the phosphor of the 5'-splice site. The second step follows where the hydroxyl of the 3'-end of the upstream exon attacks the phosphor at the 3'-end of the intron. The intron is liberated with a covalently attached guanosine and a functional messenger RNA is produced.The core of the nrdB group I intron was used in computer modelling. The 3D arrangement was according to a model by Kim and Cech originally proposed for the Tetrahymena group I intron. Our model consisted of three helices where a binding site for the cosubstrate guanosine and inhibitor arginine was located between the helix P1 and helix P7. Molecular dynamics simulation forced the cosubstrate guanosine closer to the splice site at the 5'-end of the intron.2'-Amino-2'-deoxyguanosine was found to be a cosubstrate of the nrdB group I intron self-splicing reaction. The catalytic efficiency for 2'-amino-2'-deoxyguanosine was 22-fold lower compared to guanosine catalysed self-splicing in the reaction of the wild type premessenger RNA of the nrdB gene. The wild type premessenger RNA was reconstructed and the splicing of the new shortened nrdB pre-mRNA was investigated by analysing splicing products, and the pH dependence for guanosine and 2'-amino-2'-deoxyguanosine. The study supported that the self-splicing reaction of the shortened nrdB pre-mRNA was amenable to kinetic analysis and that the chemical cleavage step was monitored under certain reaction conditions.The 2'-amino-2'-deoxyguanosine was susbsequently used to probe metal ion interaction with the cosubstrate in group I intron self-splicing. The rate of the cleavage step, catalysed by 2'-amino-2'-deoxyguanosine or guanosine, was examined in presence of different divalent metal ions or mixtures thereof. It could be demonstrated that 2'-amino-2'-deoxyguanosine becomes a better cosubstrate in presence of Mn2+ or Mg2+ and Zn2+ compared to Mg2+ alone. The increase in rate for 2'-amino-2'-deoxyguanosine was 35-fold for mixtures of Mg2+ and Zn2+ while the rate for guanosine was not affected. The results are the first experimental evidence that support the proposed two-metal-ion mechanism for group I introns.