Thermotoga maritima ribonuclease III. Characterization of thermostable biochemical behavior and analysis of conserved base pairs that function as reactivity epitopes for the Thermotoga 23S rRNA precursor.
Thermotoga maritima ribonuclease III. Characterization of thermostable biochemical behavior and analysis of conserved base pairs that function as reactivity epitopes for the Thermotoga 23S rRNA precursor.
复制标题
海栖热袍菌核糖核酸酶 III。
DOI：
10.1021/bi100930u
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发表时间：
2010
期刊：
影响因子：
2.9
通讯作者：
Nicholson,AllenW
中科院分区：
文献类型：
--
作者：
Nathania,Lilian;Nicholson,AllenW
核糖核酸酶 III 对双链 (ds) RNA 的切割是细菌 rRNA 成熟中保守的早期步骤。 RNase III 切割 dsRNA 机制的研究主要集中在来自嗜温菌（例如大肠杆菌）的酶上。相比之下，无论是极端微生物RNA酶III的催化特性还是其同源底物的结构和反应性都没有被描述。使用纯化的重组酶分析了超嗜热细菌 Thermotoga maritima 的 RNase III 生化行为。 maritima(Tm) RNase III 催化活性表现出 ∼40−70 °C 的广泛最佳温度范围，在 95 °C 时具有显着活性。 Tm-RNase III 底物裂解最好由 ≥1 mM 浓度的 Mg2+ 支持。 Mn2+、Co2+ 和 Ni2+ 也支持活性，但效率较低。该酶在 pH 8 和~50−80 mM 盐浓度下发挥最佳作用。包含 16S 和 23S pre-rRNA 茎序列的小 RNA 发夹可被 Tm-RNase III 在与成熟 rRNA 的直接前体的体内产生一致的位点有效切割。对前 23S 底物变体的分析揭示了反应性对近端盒 (pb) 中碱基对 (bp) 序列的依赖性，该位点是蛋白质接触位点，充当大肠杆菌 (Ec) RNase III 底物的阳性识别决定簇。反应性对 pb 序列的依赖性与用 Ec-RNase III 底物观察到的相似。事实上，Tm-RNase III 以相同的特异性裂解 Ec-RNase III 底物，并被抗决定簇 bp 抑制，而抗决定簇 bp 也抑制 Ec-RNase III。这些结果表明，细菌 RNase III 底物反应性的正决定因素和负决定因素在广泛的系统发育距离上保持保守。
The cleavage of double-stranded (ds) RNA by ribonuclease III is a conserved early step in bacterial rRNA maturation. Studies on the mechanism of dsRNA cleavage by RNase III have focused mainly on the enzymes from mesophiles such asEscherichia coli. In contrast, neither the catalytic properties of extremophile RNases III nor the structures and reactivities of their cognate substrates have been described. The biochemical behavior of RNase III of the hyperthermophilic bacteriumThermotoga maritimawas analyzed using purified recombinant enzyme.T. maritima(Tm) RNase III catalytic activity exhibits a broad optimal temperature range of ∼40−70 °C, with significant activity at 95 °C. Tm-RNase III cleavage of substrate is optimally supported by Mg2+at ≥1 mM concentrations. Mn2+, Co2+, and Ni2+also support activity but with reduced efficiencies. The enzyme functions optimally at pH 8 and ∼50−80 mM salt concentrations. Small RNA hairpins that incorporate the 16S and 23S pre-rRNA stem sequences are efficiently cleaved by Tm-RNase III at sites that are consistent with productionin vivoof the immediate precursors to the mature rRNAs. Analysis of pre-23S substrate variants reveals a dependence of reactivity on the base-pair (bp) sequence in the proximal box (pb), a site of protein contact that functions as a positive recognition determinant forEscherichia coli(Ec) RNase III substrates. The dependence of reactivity on the pb sequence is similar to that observed with Ec-RNase III substrates. In fact, Tm-RNase III cleaves an Ec-RNase III substrate with identical specificity and is inhibited by antideterminant bp that also inhibit Ec-RNase III. These results indicate the conservation, across a broad phylogenetic distance, of positive and negative determinants of reactivity of bacterial RNase III substrates.