16S ribosomal RNA pseudouridine synthase RsuA of Escherichia coli:: Deletion, mutation of the conserved Asp102 residue, and sequence comparison among all other pseudouridine synthases
16S ribosomal RNA pseudouridine synthase RsuA of Escherichia coli:: Deletion, mutation of the conserved Asp102 residue, and sequence comparison among all other pseudouridine synthases
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DOI：
10.1017/s1355838299990167
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发表时间：
1999-06-01
期刊：
影响因子：
4.5
通讯作者：
Ofengand, J
中科院分区：
文献类型：
--
作者：
Conrad, J;Niu, LH;Ofengand, J
大肠杆菌16 S核糖体RNA中将U 516转化为假尿苷的假尿苷合成酶RsuA基因在MG 1655和BL 21/DE 3中缺失，该基因的缺失导致两种细胞系中假尿苷516的特异性缺失，而质粒上的反式基因的替换恢复了假尿苷。因此，rsuA是大肠杆菌中唯一的基因，在24 ℃、37 ℃或42 ℃下，在丰富培养基或基本培养基中对rsuA（-）MG 1655的生长速率没有影响，BL 21/DE 3 rsuA的质粒拯救-使用含有天冬氨酸102被天冬酰胺或苏氨酸取代的rsuA基因的pET 15 b的菌株证明，两种突变体在体内都没有活性，该结果支持了这种天冬氨酸的作用，位于该基因的独特GRLD序列中，在合酶的催化中心，在菌株BL 21/BL 22中诱导野生型和两种突变体的丝氨酸蛋白酶。来自pET 15 b中基因的DE 3产生了几乎等量的所有三种蛋白质的强过表达，表明突变不影响蛋白质在体内的产生，因此活性的粘性不是由于不能产生基因产品天冬氨酸102存在于许多假尿苷激酶的保守基序中，评估了该基序在自然界中的保守性和分布。
The gene for RsuA, the pseudouridine synthase that converts U516 to pseudouridine in 16S ribosomal RNA of Escherichia coli, has been deleted in strains MG1655 and BL21/DE3, Deletion of this gene resulted in the specific loss of pseudouridine516 in both cell lines, and replacement of the gene in trans on a plasmid restored the pseudouridine, Therefore, rsuA is the only gene in E, coli with the ability to produce a protein capable of forming pseudouridine516, There was no effect on the growth rate of rsuA(-) MG1655 either in rich or minimal medium at either 24, 37, or 42 degrees C, Plasmid rescue of the BL21/DE3 rsuA- strain using pET15b containing an rsuA gene with aspartate 102 replaced by asparagine or threonine demonstrated that neither mutant was active in vivo, This result supports a role for this aspartate, located in a unique GRLD sequence in this gene, at the catalytic center of the synthase, Induction of wild-type and the two mutant synthases in strain BL21/DE3 from genes in pET15b yielded a strong overexpression of all three proteins in approximately equal amounts showing that the mutations did not affect production of the protein in vivo and thus that the tack of activity was not due to a failure to produce a gene product. Aspartate102 is found in a conserved motif present in many pseudouridine synthases, The conservation and distribution of this motif in nature was assessed.