Huang L, Ku J, Pookanjanatavip M, Gu X, Wang D, Greene P J, Santi D V
Department of Biochemistry and Biophysics, University of California, San Francisco 94143-0448, USA.
Biochemistry. 1998 Nov 10;37(45):15951-7. doi: 10.1021/bi981002n.
Several putative Escherichia coli pseudouridine (Psi) synthases have been identified by iterative searching of genomic databases for ORFs homologous to known Psi synthases [Gustafsson et al. (1996) Nucleic Acids Res. 24, 3756-3762]. Of these, yceC and yfiI were proposed to encode Psi synthases which modify 23S rRNA. In the present work, yceC and yfiI were cloned and overexpressed in E. coli, and the encoded enzymes, YceC and YfiI, were purified to homogeneity. Both proteins converted Urd residues of rRNA to Psi, thus confirming their identities as Psi synthases. However, in in vitro experiments both enzymes extensively modified Urd residues of both 23S rRNA and 16S rRNA. Gene-disruption of yceCresulted in the absence of Psi modification at positions U955, 2504, and 2580 of 23S RNA, thus identifying these sites as in vivo targets for YceC. Likewise, yfiI disruption resulted in the absence of Psi modification at positions U1911, 1917, and possibly 1915 of 23S RNA. Disruption of yceC did not affect the growth under the conditions tested, whereas yfiI-disrupted cells showed a dramatic decrease in growth rate. Since YceC and YfiI hypermodify RNA in vitro, factors in addition to ribonucleotide sequence must contribute to the in vivo specificity of these enzymes.
通过对基因组数据库进行迭代搜索，寻找与已知假尿苷（Ψ）合酶同源的开放阅读框（ORF），已鉴定出几种假定的大肠杆菌假尿苷合酶[古斯塔夫松等人（1996年）《核酸研究》24卷，3756 - 3762页]。其中，yceC和yfiI被认为编码修饰23S rRNA的Ψ合酶。在本研究中，yceC和yfiI在大肠杆菌中被克隆并过表达，编码的酶YceC和YfiI被纯化至同质。两种蛋白质都将rRNA的尿苷（Urd）残基转化为Ψ，从而证实了它们作为Ψ合酶的身份。然而，在体外实验中，两种酶都广泛修饰了23S rRNA和16S rRNA的Urd残基。yceC的基因破坏导致23S RNA的U955、2504和2580位点没有Ψ修饰，因此确定这些位点是YceC在体内的作用靶点。同样，yfiI的破坏导致23S RNA的U1911、1917以及可能的1915位点没有Ψ修饰。yceC的破坏在测试条件下不影响生长，而yfiI破坏的细胞显示生长速率急剧下降。由于YceC和YfiI在体外对RNA进行过度修饰，除核糖核苷酸序列外的其他因素必定对这些酶的体内特异性有影响。
