Gutgsell N S, Del Campo M, Raychaudhuri S, Ofengand J
Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, Florida 33101, USA.
RNA. 2001 Jul;7(7):990-8. doi: 10.1017/s1355838201000243.
This laboratory previously showed that truncation of the gene for RluD, the Escherichia coli pseudouridine synthase responsible for synthesis of 23S rRNA pseudouridines 1911, 1915, and 1917, blocks pseudouridine formation and inhibits growth. We now show that RluD mutants at the essential aspartate 139 allow these two functions of RluD to be separated. In vitro, RluD with aspartate 139 replaced by threonine or asparagine is completely inactive. In vivo, the growth defect could be completely restored by transformation of an RluD-inactive strain with plasmids carrying genes for RluD with aspartate 139 replaced by threonine or asparagine. Pseudouridine sequencing of the 23S rRNA from these transformed strains demonstrated the lack of these pseudouridines. Pseudoreversion, which has previously been shown to restore growth without pseudouridine formation by mutation at a distant position on the chromosome, was not responsible because transformation with empty vector under identical conditions did not alter the growth rate.
本实验室先前表明，截短负责合成23S rRNA假尿苷1911、1915和1917的大肠杆菌假尿苷合酶RluD的基因，会阻断假尿苷的形成并抑制生长。我们现在表明，关键天冬氨酸139位点发生突变的RluD突变体可使RluD的这两种功能分离。在体外，天冬氨酸139被苏氨酸或天冬酰胺取代的RluD完全无活性。在体内，通过用携带天冬氨酸139被苏氨酸或天冬酰胺取代的RluD基因的质粒转化RluD无活性菌株，可完全恢复生长缺陷。对这些转化菌株的23S rRNA进行假尿苷测序表明，这些假尿苷缺失。先前已表明，假回复突变可通过染色体上远处位置的突变恢复生长而不形成假尿苷，但这种情况并不成立，因为在相同条件下用空载体转化不会改变生长速率。
