Crombie T, Boyle J P, Coggins J R, Brown A J
Department of Molecular and Cell Biology, University of Aberdeen, Marischal College, Scotland.
Eur J Biochem. 1994 Dec 1;226(2):657-64. doi: 10.1111/j.1432-1033.1994.tb20093.x.
The yeast TRP3 gene encodes a bifunctional protein with anthranilate synthase II and indoleglycerol-phosphate synthase activities. Replacing ten consecutive non-preferred codons in the indoleglycerol-phosphate synthase region of the TRP3 gene with synonymous preferred codons (to create the TRP3pr gene; translational pause replaced) causes a 1.5-fold reduction in relative indoleglycerol-phosphate synthase activity [Crombie, T., Swaffield, J.C. & Brown, A.J.P. (1992) J. Mol. Biol. 228, 7-12]. Here, we report that both the anthranilate synthase II and indoleglycerol-phosphate synthase domains are affected to similar extents when the translational pause is removed. Also, structural modelling of the yeast indoleglycerol-phosphate synthase domain against the X-ray crystal structure of indoleglycerol-phosphate synthase from Escherichia coli indicates that the translational pause lies in a region of structural divergence between similar structures. To probe the role of cytoplasmic heat-shock protein 70 (Hsp 70) chaperones in Trp3 protein folding, anthranilate synthase and indoleglycerol-phosphate synthase activities were measured in ssa and ssb mutants. Neither indoleglycerol-phosphate synthase nor anthranilate synthase were affected significantly in the ssb mutant. However, depletion of Hsp70 proteins encoded by the SSA genes led to decreased anthranilate synthase and indoleglycerol-phosphate synthase activities from the TRP3 gene, suggesting that both domains depend to some extent upon the SSA chaperone family. The data are consistent with roles for both the translational pause and Ssa chaperones in Trp3 protein folding in vivo.
酵母TRP3基因编码一种具有邻氨基苯甲酸合酶II和吲哚甘油磷酸合酶活性的双功能蛋白。用同义的优先密码子替换TRP3基因的吲哚甘油磷酸合酶区域中的10个连续的非优先密码子（以创建TRP3pr基因；翻译暂停被消除）会导致吲哚甘油磷酸合酶相对活性降低1.5倍[克朗比，T.，斯沃菲尔德，J.C. & 布朗，A.J.P.（1992年）《分子生物学杂志》228卷，7 - 12页]。在此，我们报告当翻译暂停被消除时，邻氨基苯甲酸合酶II和吲哚甘油磷酸合酶结构域受到的影响程度相似。此外，根据大肠杆菌吲哚甘油磷酸合酶的X射线晶体结构对酵母吲哚甘油磷酸合酶结构域进行的结构建模表明，翻译暂停位于相似结构之间的结构差异区域。为了探究细胞质热休克蛋白70（Hsp 70）伴侣蛋白在Trp3蛋白折叠中的作用，在ssa和ssb突变体中测量了邻氨基苯甲酸合酶和吲哚甘油磷酸合酶的活性。在ssb突变体中，吲哚甘油磷酸合酶和邻氨基苯甲酸合酶均未受到显著影响。然而，由SSA基因编码的Hsp70蛋白的缺失导致TRP3基因的邻氨基苯甲酸合酶和吲哚甘油磷酸合酶活性降低，这表明两个结构域在一定程度上都依赖于SSA伴侣蛋白家族。这些数据与翻译暂停和Ssa伴侣蛋白在体内Trp3蛋白折叠中的作用一致。
