Valenzuela L, Ballario P, Aranda C, Filetici P, González A
Departamento de Genética Molecular, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Mexico City 04510, Mexico.
J Bacteriol. 1998 Jul;180(14):3533-40. doi: 10.1128/JB.180.14.3533-3540.1998.
Saccharomyces cerevisiae glutamate synthase (GOGAT) is an oligomeric enzyme composed of three 199-kDa identical subunits encoded by GLT1. In this work, we analyzed GLT1 transcriptional regulation. GLT1-lacZ fusions were prepared and GLT1 expression was determined in a GDH1 wild-type strain and in a gdh1 mutant derivative grown in the presence of various nitrogen sources. Null mutants impaired in GCN4, GLN3, GAT1/NIL1, or UGA43/DAL80 were transformed with a GLT1-lacZ fusion to determine whether the above-mentioned transcriptional factors had a role in GLT1 expression. A collection of increasingly larger 5' deletion derivatives of the GLT1 promoter was constructed to identify DNA sequences that could be involved in GLT1 transcriptional regulation. The effect of the lack of GCN4, GLN3, or GAT1/NIL1 was also tested in the pertinent 5' deletion derivatives. Our results indicate that (i) GLT1 expression is negatively modulated by glutamate-mediated repression and positively regulated by Gln3p- and Gcn4p-dependent transcriptional activation; (ii) two cis-acting elements, a CGGN15CCG palindrome and an imperfect poly(dA-dT), are present and could play a role in GLT1 transcriptional activation; and (iii) GLT1 expression is moderately regulated by GCN4 under amino acid deprivation. Our results suggest that in a wild-type strain grown on ammonium, GOGAT constitutes an ancillary pathway for glutamate biosynthesis.
酿酒酵母谷氨酸合酶（GOGAT）是一种寡聚酶，由GLT1编码的三个199 kDa的相同亚基组成。在这项工作中，我们分析了GLT1的转录调控。制备了GLT1 - lacZ融合体，并在存在各种氮源的情况下，在GDH1野生型菌株和gdh1突变体衍生物中测定了GLT1的表达。用GLT1 - lacZ融合体转化在GCN4、GLN3、GAT1/NIL1或UGA43/DAL80中受损的缺失突变体，以确定上述转录因子是否在GLT1表达中起作用。构建了一系列GLT1启动子越来越大的5'缺失衍生物，以鉴定可能参与GLT1转录调控的DNA序列。还在相关的5'缺失衍生物中测试了缺乏GCN4、GLN3或GAT1/NIL1的影响。我们的结果表明：（i）GLT1表达受到谷氨酸介导的抑制的负调控，并受到Gln3p和Gcn4p依赖性转录激活的正调控；（ii）存在两个顺式作用元件，一个CGGN15CCG回文序列和一个不完美的聚（dA - dT），它们可能在GLT1转录激活中起作用；（iii）在氨基酸剥夺条件下，GLT1表达受到GCN4的适度调控。我们的结果表明，在以铵为生长底物的野生型菌株中，GOGAT构成了谷氨酸生物合成的辅助途径。
