SEQUENCE AND STRUCTURE REQUIREMENTS FOR DROSOPHILA TRANSFER-RNA 5'-END AND 3'-END PROCESSING
SEQUENCE AND STRUCTURE REQUIREMENTS FOR DROSOPHILA TRANSFER-RNA 5'-END AND 3'-END PROCESSING
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DOI：
10.1074/jbc.270.32.18903
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发表时间：
1995-08-11
影响因子：
4.8
通讯作者：
KOLLA, S
中科院分区：
文献类型：
--
作者：
LEVINGER, L;VASISHT, V;KOLLA, S
真核生物 tRNA 的 5'- 和 3'- 末端分别由核酸内切酶 RNase P 和 3'-tRNase 加工。我们制备了这两种酶的底物，从果蝇提取物中分离出活性，并设计了变体 tRNA 以评估序列和结构对加工的影响。突变以类似的方式影响这些反应。因此，RNase P 和 3'-tRNase 可能需要相似的底物结构来维持催化配合。 RNase P 比 3'-tRNase 对底物取代更敏感。在四个茎中的三个中，一种取代阻止了两种加工反应，而相反的一种则影响较小；反密码子茎替换几乎不影响加工，而旨在恢复碱基配对的双替换也将加工恢复到野生型速率。结构探测表明，tRNA 错误折叠有时与加工减少同时发生。在其他情况下，加工抑制可能是由于特定的不利茎并置导致局部螺旋变形所致。单个 T 环替换会破坏三级 D-T 环相互作用并减少处理。因此，我们开始在全局、局部和三级结构水平上绘制 tRNA 加工决定因素。
Eukaryotic tRNAs are processed at their 5'- and 3'-ends by the endonucleases RNase P and 3'-tRNase, respectively, We have prepared substrates for both enzymes, separated the activities from a Drosophila extract, and designed variant tRNAs to assess the effects of sequence and structure on processing. Mutations affect these reactions in similar ways; thus, RNase P and 3'-tRNase probably require similar substrate structures to maintain the catalytic fit. RNase P is more sensitive to substrate substitutions than 3'-tRNase. In three of the four stems, one substitution prevents both processing reactions while the opposite one has less effect; anticodon stem substitutions hardly affect processing, and double substitutions intended to restore base pairing also restore processing to the wild type rate.Structure probing suggests that tRNA misfolding sometimes coincides with reduced processing. In other cases, processing inhibition probably results from specific unfavorable stem appositions leading to local helix deformation. A single T loop substitution disrupts the tertiary D-T loop interaction and reduces processing. We have thus begun mapping tRNA processing determinants on the global, local, and tertiary structure levels.