The effects of matrices of paired substitutions in mid-acceptor stem on Drosophila tRNA(His) structure and end-processing.
The effects of matrices of paired substitutions in mid-acceptor stem on Drosophila tRNA(His) structure and end-processing.
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中部受体茎中成对取代矩阵对果蝇 tRNA(His) 结构和末端加工的影响。
DOI：
10.1006/jmbi.2000.4162
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发表时间：
2000
期刊：
影响因子：
--
通讯作者：
Levinger,L
中科院分区：
文献类型：
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作者：
Mohan,A;Levinger,L
末端成熟反应是真核生物前体tRNA加工过程中早期的重要步骤，在此过程中，前体tRNA的5′端前导序列和3′端尾序列分别被RNase P和3′-tRNase去除。末端加工酶可能会接触tRNA的受体茎，因为它接近两个切割位点。我们构建了果蝇tRNAHis中间受体茎3/70和4/69 nt处的成对取代矩阵，并分析了它们被果蝇RNase P和3′-tRNase加工的能力。与我们早期对D/T环加工矩阵的研究雅阁，我们发现tRNA末端加工酶对序列变化的反应不同。3′-tRNase比RNase P有更多的加工缺陷，4/69处的替换比3/70处的替换减少了更多的加工。我们使用结构探测核酸酶评估了tRNA折叠，并研究了Km和Vmax对所选变体的加工效率的贡献。在一个取代（C3 A）中，错误折叠与加工缺陷相关。在另一个（C69 A）中，结构的破坏似乎横向传递到受体茎的两端。RNase P对C69 A的不良加工完全是由于VMax的降低，但对于3′-tRNase，这是由于KM的增加。
End-maturation reactions, in which the 5′ end leader and 3′ end trailer of precursor tRNA are removed by RNase P and 3′-tRNase, respectively, are early, essential steps in eukaryotic precursor tRNA processing. End-processing enzymes may be expected to contact the acceptor stem of tRNA due to its proximity to both cleavage sites. We constructed matrices of pair-wise substitutions in mid-acceptor stem at nt 3/70 and 4/69 of Drosophila tRNAHisand analyzed their ability to be processed by Drosophila RNase P and 3′-tRNase. In accord with our earlier study of D/T loop processing matrices, we find that tRNA end processing enzymes respond to sequence changes differently. More processing defects were observed with 3′-tRNase than with RNase P, and substitutions at 4/69 reduced processing more than those at 3/70. We evaluated tRNA folding using structure probing nucleases and investigated the contribution of KMand VMaxto the processing efficiency of selected variants. In one substitution (C3A), mis-folding correlates with processing defects. In another (C69A), a disruption of structure appears to be transmitted laterally to both ends of the acceptor stem. Poor processing of C69A by RNase P is due entirely to a reduction in VMax, but for 3′-tRNase, it is due to an increase in KM.