Wrzesinski J, Bakin A, Nurse K, Lane B G, Ofengand J
Roche Institute of Molecular Biology, Roche Research Center, Nutley, New Jersey 07110, USA.
Biochemistry. 1995 Jul 11;34(27):8904-13. doi: 10.1021/bi00027a043.
Pseudouridine (psi) is commonly found in both small and large subunit ribosomal RNAs of prokaryotes and eukaryotes. In Escherichia coli small subunit RNA, there is only one psi, at position 516, in a region of the RNA known to be involved in codon recognition [Bakin et al. (1994) Nucleic Acids Res. 22, 3681-3684]. To assess the function of this single psi residue, the enzyme catalyzing its formation was purified and cloned. The enzyme contains 231 amino acids and has a calculated molecular mass of 25,836 Da. It converts U516 in E. coli 16S RNA transcripts into psi but does not modify any other position in this RNA. It does not react with free unmodified 16S RNA at all, and only poorly with 30S particles containing unmodified RNA. The preferred substrate is an RNA fragment from residues 1 to 678 which has been complexed with 30S ribosomal proteins. The yield varied from 0.6 to 1.0 mol of psi/mol of RNA, depending on the preparation. Free RNA(1-678) was inactive, as was RNA(1-526) and the RNP particle made from it. 23S RNA and tRNAVal transcripts were also inactive. These results suggest that psi formation in vivo occurs at an intermediate stage of 30S assembly. The gene is located at 47.1 min immediately 5' to, and oriented in the same direction as, the bicyclomycin resistance gene. The gene was cloned behind a (His)6 leader for affinity purification. Virtually all of the overexpressed protein was found in inclusion bodies but could be purified to homogeneity on a Ni2+(-) containing resin. Over 200 mg of pure protein could be obtained from a liter of cell culture. Amino acid sequence comparison revealed the existence of a gene in Bacillus subtilis with a similar sequence, and psi sequence analysis established that B. subtilis has the equivalent of psi 516 in its small subunit rRNA. On the other hand, no common sequence motifs could be detected among this enzyme and the two tRNA psi synthases which have been cloned up to now.
假尿苷（ψ）普遍存在于原核生物和真核生物的小亚基和大亚基核糖体RNA中。在大肠杆菌小亚基RNA中，已知参与密码子识别的RNA区域（位置516处）仅有一个ψ [贝金等人（1994年），《核酸研究》22卷，3681 - 3684页]。为评估这个单一ψ残基的功能，催化其形成的酶被纯化并克隆。该酶含有231个氨基酸，计算分子量为25,836道尔顿。它将大肠杆菌16S RNA转录本中的U516转化为ψ，但不修饰该RNA中的任何其他位置。它根本不与游离的未修饰16S RNA反应，与含有未修饰RNA的30S颗粒反应也很弱。优选的底物是与30S核糖体蛋白复合的、来自1至678位残基的RNA片段。产率根据制备情况在每摩尔RNA产生0.6至1.0摩尔ψ之间变化。游离的RNA(1 - 678)无活性，RNA(1 - 526)及其制成的核糖核蛋白颗粒也无活性。23S RNA和tRNAVal转录本也无活性。这些结果表明，体内ψ的形成发生在30S组装的中间阶段。该基因位于紧邻双环霉素抗性基因5'端47.1分钟处，且与双环霉素抗性基因方向相同。该基因被克隆在一个（His）6前导序列之后用于亲和纯化。几乎所有过表达的蛋白质都存在于包涵体中，但可在含Ni2 +的树脂上纯化至同质。从一升细胞培养物中可获得超过200毫克的纯蛋白。氨基酸序列比较显示枯草芽孢杆菌中存在一个序列相似的基因，且ψ序列分析确定枯草芽孢杆菌小亚基rRNA中有相当于ψ516的序列。另一方面，在该酶与目前已克隆的两种tRNA ψ合成酶之间未检测到共同的序列基序。
