净化、克隆和516年16 s RNA假尿苷合成酶的属性从大肠杆菌。
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Wrzesinski J, Bakin,护士K, Lane BG Ofengand J
净化、克隆和516年16 s RNA假尿苷合成酶的属性从大肠杆菌。
生物化学。1995年7月11日,34(27):8904 - 13所示。
- PubMed ID
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7612632 (在PubMed]
- 文摘
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假尿苷(psi)是常见的在这两个小型和大型亚基原核生物和真核生物的核糖体rna。在大肠杆菌小亚基RNA, psi,只有一个在516的位置,在一个地区已知的RNA参与密码子识别[Bakin et al。(1994)核酸研究》22日3681 - 3684]。评估这个单一的函数ψ残渣,酶催化净化和克隆形成。酶含有231个氨基酸,分子质量25836 Da计算。它转换U516大肠杆菌16 s RNA转录到psi但不修改任何其他位置的RNA。它不与自由修改的16 s RNA,反应,只差30年代的颗粒包含修改的RNA。首选的衬底的RNA片段残留1到678已与30年代核糖体蛋白质。psi的产量变化从0.6到1.0摩尔/摩尔的RNA,这取决于准备。自由RNA(1 - 678)是不活跃的,就像RNA(1 - 526)和RNP粒子制成的。23 s RNA和tRNAVal成绩单也不活跃。 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.