Deletion analysis of domain independence in the TRP1 gene product of Neurospora crassa.
The trifunctional TRP1 gene from Neurospora crassa (N-TRP1) was subcloned into the yeast-Escherichia coli shuttle vector YEp13 and expressed in Saccharomyces cerevisiae. The three activities of the N-TRP1 gene product were detected in yeast mutants that lacked either N-(5'-phosphoribosyl) anthranilate (PRA) isomerase or both the glutamine amidotransferase function of anthranilate synthase and indole-3-glycerol phosphate (InGP) synthase. The protein was detected on immunoblots only as the full length 83 kda product indicating that the trifunctional gene product was expressed in yeast primarily in a fully active, undegraded form. By placing the subcloned N-TRP1 gene under the control of the inducible PHO5 promoter from yeast, the expression of all three activities was increased to more than ten fold that of wild-type yeast and the overproduced protein could be visualized by SDS-polyacrylamide gel electrophoresis of crude extract and Coomassie Blue staining. Using the expression system described the effect of selective deletion of regions of the coding sequence of the N-TRP1 gene on expression of the three activities was tested. Expression of either the F- or C-domains, catalyzing respectively the PRA isomerase or InGP synthase activities, did not depend on the presence of the other domain in the active polypeptide. Furthermore, normal dimer formation occurred with a protein active for InGP synthase in a deletion derivative lacking most of the PRA isomerase domain, ruling out the hypothesis that interaction between the active site regions for PRA isomerase and InGP synthase accounted for dimer formation in the trifunctional product.
Walker
M S
MS
Department of Biochemistry and Molecular Biology, University of Texas Medical School, Houston 77225.
DeMoss
J A
JA
D000877:Anthranilate Phosphoribosyltransferase / Q000235:genetics* / Q000378:metabolism; D000878:Anthranilate Synthase / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D002845:Chromatography; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal; D005816:Genetic Complementation Test; D015151:Immunoblotting; D007209:Indole-3-Glycerol-Phosphate Synthase / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009492:Neurospora crassa / Q000201:enzymology / Q000235:genetics*; D019884:Nitrogenous Group Transferases; D011401:Promoter Regions, Genetic; D015183:Restriction Mapping; D014166:Transferases / Q000235:genetics* / Q000378:metabolism
D014166:Transferases; D000877:Anthranilate Phosphoribosyltransferase; D019884:Nitrogenous Group Transferases; D007209:Indole-3-Glycerol-Phosphate Synthase; D000878:Anthranilate Synthase; C020655:anthranilate synthase, glutamine amidotransferase subunit