title: Preceding hydrophobic and beta-branched amino acids attenuate splicing by the CnePRP8 intein.
abstract: As the Cne PRP8 intein is active and exists in an essential gene of an important fungal pathogen, inhibitors of splicing and assays for intein activity are of interest. The self-splicing activity of Cne PRP8, the intein from the Prp8 gene of Cryptococcus neoformans, was assessed in different heterologous fusion proteins expressed in Escherichia coli. Placement of a putatively inactive variant of the intein adjacent to the alpha-complementation peptide abolished the peptide's ability to restore beta-galactosidase activity, while an active variant allowed complementation. This alpha-complementation peptide therefore provides a facile assay of splicing which can be used to test potential inhibitors. When placed between two heterologous protein domains, splicing was impaired by a beta-branched amino acid immediately preceding the intein, while splicing occurred only with a hydroxyl or thiol immediately following the intein. Both these assays sensitively report impairment of splicing and provide information on how context constrains the splicing ability of Cne PRP8.
authors.affiliation: Department of Biochemistry, University of Otago, PO Box 56, Dunedin, New Zealand.
authors.forename: Esther J
authors.initials: EJ
authors.lastname: Pearl
authors.forename: Annika A M
authors.initials: AA
authors.lastname: Bokor
authors.forename: Margi I
authors.initials: MI
authors.lastname: Butler
authors.forename: Russell T M
authors.initials: RT
authors.lastname: Poulter
authors.forename: Sigurd M
authors.initials: SM
authors.lastname: Wilbanks
mesh_terms: D000596:Amino Acids / Q000235:genetics / Q000378:metabolism*; D001681:Biological Assay; D003455:Cryptococcus neoformans / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D005816:Genetic Complementation Test; D057927:Hydrophobic and Hydrophilic Interactions; D047668:Inteins / Q000502:physiology*; D019154:Protein Splicing / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
chemical_list: D000596:Amino Acids; D005656:Fungal Proteins; D011993:Recombinant Fusion Proteins; D001616:beta-Galactosidase