Modified constructs of the tRNA TΨC domain to probe substrate conformational requirements of m1A58 and m5U54 tRNA methyltransferases
Modified constructs of the tRNA TΨC domain to probe substrate conformational requirements of m1A58 and m5U54 tRNA methyltransferases
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DOI：
10.1093/nar/28.6.1374
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发表时间：
2000-03-15
影响因子：
14.9
通讯作者：
Agris, PF
中科院分区：
文献类型：
--
作者：
Sengupta, R;Vainauskas, S;Agris, PF
tRNA的T Psi C茎环（TSL）含有高度保守的核苷修饰，m（5）C（49），T-54，Psi（55）和m（1）A（58）。U-54被m（5）U（54）甲基转移酶（RUMT）甲基化为m（5）U（T）; A（58）被m（1）A（58）tRNA甲基转移酶（RAMT）甲基化为m（1）A，RUMT识别并甲基化最小的TSL十七聚体，RAMT先前已报道识别并甲基化tRNA分子的3 '-一半。我们报告，RAMT可以识别和甲基化的TSL十七聚体。为了更好地理解RAMT和RUMT对TSL构象的敏感性，我们设计并合成了各种修饰的TSL构建体，这些构建体具有改变的局部构象和稳定性。合成具有天然修饰（T-54和Psi（55））、在非天然位置处天然存在的修饰（m（5）C（60））、改变的糖皱褶（dU（54）和/或dU（55））或具有破坏的U形转弯相互作用（m（1）Psi（55）或m（1）m（3）Psi（55））的TSL。未修饰的十七聚体TSL是RAMT和RUMT两者的底物。T-54的存在增加了TSL的热稳定性，并显著降低了对底物的RAMT活性。发现U-54周围的局部构象是RAMT和RUMT活性的重要决定因素。
The T Psi C stem and loop (TSL) of tRNA contains highly conserved nucleoside modifications, m(5)C(49), T-54, Psi(55) and m(1)A(58). U-54 is methylated to m(5)U (T) by m(5)U(54) methyltransferase (RUMT); A(58) is methylated to m(1)A by m(1)A(58) tRNA methyltransferase (RAMT), RUMT recognizes and methylates a minimal TSL heptadecamer and RAMT has previously been reported to recognize and methylate the 3'-half of the tRNA molecule. We report that RAMT can recognize and methylate a TSL heptadecamer. To better understand the sensitivity of RAMT and RUMT to TSL conformation, we have designed and synthesized variously modified TSL constructs with altered local conformations and stabilities. TSLs were synthesized with natural modifications (T-54 and Psi(55)), naturally occurring modifications at unnatural positions (m(5)C(60)), altered sugar puckers (dU(54) and/or dU(55)) or with disrupted U-turn interactions (m(1)Psi(55) or m(1)m(3)Psi(55)), The unmodified heptadecamer TSL was a substrate of both RAMT and RUMT. The presence of T-54 increased thermal stability of the TSL and dramatically reduced RAMT activity toward the substrate, Local conformation around U-54 was found to be an important determinant for the activities of both RAMT and RUMT.