Functional and sequence analysis of splicing defective nrdB mutants of bacteriophage T4 reveal new bases and a new sub-domain required for group I intron self-splicing.
The nrdB gene of bacteriophage T4 codes for the small subunit of ribonucleotide reductase and contains a 598 nuclelotide group 1 self splicing intron. In order to study the functional domains for self-splicing of this intron, 23 nrdB splicing defective intron mutants were analyzed for both sequence and functional changes. These mutants cluster towards the ends in regions of conserved structural elements of the intron. These 23 mutants have single base changes at 14 different sites. Interestingly 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 structure. The mutation sites in pairing regions P3, P7, P8, P9 and between P6[3'] and P7[5'] are identical to changes found in the well studied 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 this pairing, thus defining a new sub-domain essential for RNA splicing. Like the td intron, the mutation site in P9 of the nrdB intron is a hot spot for mutations, but unlike td, the nrdB intron does not show a mutational hot spot in the P6[5'] region. Our molecular dissection of the 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 selection, cleavage and exon ligation.
Lal
S K
SK
Virology Group, International Centre for Genetic Engineering and Biotechnology, New Delhi, India. sunil@icgeb.trieste.it
Hall
D H
DH
D017122:Bacteriophage T4 / Q000201:enzymology / Q000235:genetics*; D001483:Base Sequence; D017931:DNA Primers; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005814:Genes, Viral; D007438:Introns; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D016366:Open Reading Frames; D017354:Point Mutation; D012326:RNA Splicing; D012367:RNA, Viral / Q000737:chemistry* / Q000235:genetics; D015183:Restriction Mapping; D012264:Ribonucleotide Reductases / Q000096:biosynthesis / Q000235:genetics*; D015678:Viral Structural Proteins / Q000235:genetics
D017931:DNA Primers; D046911:Macromolecular Substances; D012367:RNA, Viral; D015678:Viral Structural Proteins; D012264:Ribonucleotide Reductases