Preceding hydrophobic and beta-branched amino acids attenuate splicing by the CnePRP8 intein |
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Authors: | Pearl Esther J Bokor Annika A M Butler Margi I Poulter Russell T M Wilbanks Sigurd M |
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Institution: | Department of Biochemistry, University of Otago, PO Box 56, Dunedin, New Zealand. |
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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. |
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