Department of Chemistry and Biochemistry, Florida State University, Tallahassee, Florida 32306, USA.
Biochemistry. 2010 Jul 27;49(29):6276-81. doi: 10.1021/bi1006699.
Box H/ACA ribonucleoprotein protein particles catalyze the majority of pseudouridylation in functional RNA. Different from stand alone pseudouridine synthases, the RNP pseudouridine synthase comprises multiple protein subunits and an RNA subunit. Previous studies showed that each subunit, regardless its location, is sensitive to the step of subunit placement at the catalytic center and potentially to the reaction status of the substrate. Here we describe the impact of chemical substitutions of target uridine on enzyme activity and structure. We found that 3-methyluridine in place of uridine inhibited its isomerization while 2'-deoxyuridine or 4-thiouridine did not. Significantly, crystal structures of an archaeal box H/ACA RNP bound with the nonreactive and the two postreactive substrate analogues showed only subtle structural changes throughout the assembly except for a conserved tyrosine and a substrate anchoring loop of Cbf5. Our results suggest a potential role of these elements and the subunit that contacts them in substrate binding and product release.
盒 H/ACA 核糖核蛋白颗粒催化大多数功能性 RNA 的假尿嘧啶化。与独立的假尿嘧啶合酶不同，RNP 假尿嘧啶合酶由多个蛋白质亚基和一个 RNA 亚基组成。以前的研究表明，每个亚基，无论其位置如何，都对亚基在催化中心的放置步骤以及底物的反应状态敏感。在这里，我们描述了靶尿嘧啶的化学取代对酶活性和结构的影响。我们发现，3-甲基尿嘧啶代替尿嘧啶抑制其异构化，而 2'-脱氧尿嘧啶或 4-硫代尿嘧啶则不抑制。重要的是，与非反应性和两种后反应性底物类似物结合的古细菌盒 H/ACA RNP 的晶体结构显示，除了保守的酪氨酸和 Cbf5 的底物锚定环外，整个组装过程中只有细微的结构变化。我们的结果表明，这些元素和与它们接触的亚基在底物结合和产物释放中可能发挥作用。
