FUNCTIONAL AND SEQUENCE-ANALYSIS OF SPLICING DEFECTIVE NRDB MUTANTS OF BACTERIOPHAGE-T4 REVEAL NEW BASES AND A NEW SUBDOMAIN REQUIRED FOR GROUP-I INTRON SELF-SPLICING

The nrdB gene of bacteriophage T4 codes for the small subunit of ribon ucleotide reductase and contains a 598 nuclelotide group 1 self splici ng intron. In order to study the functional domains for self-splicing of this intron, 23 nrdB splicing defective intron mutants were analyse d for both sequence and functional changes. These mutants cluster towa rds the ends in regions of conserved structural elements of the intron . These 23 mutants have single base changes at 14 different sites. Int erestingly two of these sites that seemed to map within the intron are actually located on the flanking exon sequences on both sides of the intron. A high frequency (4/12) of the mutation sites are in bases not thought to be base-paired in the standard model of group I intron str ucture. The mutation sites in pairing regions P3, P7, P8, P9 and betwe en P6[3'] and P7[5'] are identical to changes found in the well studie d td (encoding dTMP synthase) intron. However, five new mutation sites (S61, SL1, S29, SL11, SL196 and SL126) are unique to the nrdB intron and disrupt self-splicing. A mutation (S61) in the P7.1 pairing region is especially significant because no mutations have been found in thi s pairing, thus defining a new sub-domain essential for RNA splicing. Like the rd intron, the mutation site in P9 of the nrdB intron is a ho t spot for mutations, but unlike td, the nrdB intron does not show a m utational hot spot in the P6[5'] region. Our molecular dissection of t he nrdB intron also supports the P9.0 and P10 pairings that have been postulated to help form a complex tertiary structure required to give the RNA sequence its catalytic activity: particularly 3' splice site s election, cleavage and exon ligation.