SOLUTION STRUCTURE OF THE 3'-END OF BROME MOSAIC-VIRUS GENOMIC RNAS - CONFORMATIONAL MIMICRY WITH CANONICAL TRANSFER-RNAS
SOLUTION STRUCTURE OF THE 3'-END OF BROME MOSAIC-VIRUS GENOMIC RNAS - CONFORMATIONAL MIMICRY WITH CANONICAL TRANSFER-RNAS
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DOI：
10.1006/jmbi.1994.1010
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发表时间：
1994-01-14
影响因子：
5.6
通讯作者：
WESTHOF, E
中科院分区：
文献类型：
--
作者：
FELDEN, B;FLORENTZ, C;WESTHOF, E
用不同的化学探针和酶探针研究了雀麦花叶病毒（BMV）RNA 3′端最后201个核苷酸的构象。用硫酸二甲酯（其使A的N-1位、C的N-3位和G的N-7位甲基化）、碳二亚胺（其修饰G的N-1位和U的N-3位）和焦碳酸二乙酯（其修饰A的N-7位）探测碱基。核糖核酸酶T1、U2和S1用于未配对核苷酸的定位，核糖核酸酶V1用于配对碱基或堆叠核苷酸的定位。通过凝胶电泳检测切割或修饰位点，或者间接地通过分析经修饰的RNA的逆转录酶的引物延伸产生的DNA序列模式，或者通过在RNA的统计切割模式内直接鉴定。在此基础上，通过计算机模拟建立了RNA的原子模型，并对其立体化学进行了修正，得到了RNA的二级结构，证实了前人提出的数据，但含有额外的碱基对（A27-U32、A28-G31、G41-A134、G64-C68、U80-A99、G81-A98、G88-U91、G100-U126、U104-U125、G162-G166和A172-A191），一个新的三级长程相互作用（U103-U164）和一个小的三重螺旋构象（G41-A134）-A18和（C42-G133）-A17相互作用。新的二级结构还表明存在第二个假结，包括残基A181至A184和残基U197至U194之间的配对，在赋予BMV RNA酪氨酸基化能力的结构域之外。该模型的主要结果源于其复杂的折叠，这允许对模仿tRNA的反密码子和D环区域的结构域进行新的分配。有趣的是，茎和环区发现结构类似于反密码子臂tRNATyrdoes不包含参与氨酰化过程的酪氨酸反密码子。与经典tRNATy的结构相似性说明了BMV RNA结构与经典tRNATy之间存在的功能模拟，这允许它们被酪氨酰-tRNA合成酶有效地氨酰化。该结构模型合理化了诱变和足迹数据，这些数据已经确定了病毒RNA特定区域对其复制酶（ATP，CTP）：tRNA核苷酸转移酶和酵母酪氨酰-tRNA合成酶识别的重要性。新的折叠具有生物学意义，可以作为制定新实验的预测工具。
The conformation of the last 201 nucleotides located at the 3′-end of brome mosaic virus (BMV) RNAs was investigated in solution using different chemical and enzymatic probes. Bases were probed with dimethylsulfate (which methylates N-1 positions of A, N-3 positions of C and N-7 positions of G), a carbodiimide (which modifies N-1 positions of G and N-3 positions of U) and diethylpyrocarbonate (which modifies N-7 positions of A). Ribonucleases T1, U2and S1were used to map unpaired nucleotides and ribonuclease V1to monitor paired bases or stacked nucleotides. Cleavage or modification sites were detected by gel electrophoresis either indirectly by analyzing DNA sequence patterns generated by primer extension with reverse transcriptase of the modified RNAs or by direct identification within the statistical cleavage patterns of the RNA. On the basis of these biochemical results, an atomic model was built by computer modeling and its stereochemistry refined.The deduced secondary structure of the RNA confirms data previously proposed by others but contains additional base-pairs (A27-U32, A28-G31, G41-A134, G64-C68, U80-A99, G81-A98, G88-U91, G100-U126, U104-U125, G162-G166 and A172-A191), one new tertiary long range interaction (U103-U164) and a small triple helical conformation with (G41-A134)-A18 and (C42-G133)-A17 interactions. The new secondary structure also indicates the existence of a second pseudoknot involving pairing between residues A181 to A184 and residues U197 to U194, outside the domain conferring tyrosylation ability to BMV RNA.The main outcome from the model stems from its intricate folding, which allows a new assignment for the domains mimicking the anticodon- and D-loop regions of tRNA. Interestingly, the stem and loop region found structurally to be analogous to the anticodon arm of tRNATyrdoes not contain the tyrosine anticodon involved in the aminoacylation process. The structural analogies with canonical tRNATyrillustrate the functional mimicry existing between the BMV RNA structure and canonical tRNATyrthat allows for their efficient aminoacylation by tyrosyl-tRNA synthetase. This structural model rationalizes mutagenic and footprinting data that have established the importance of specific regions of the viral RNA for recognition by its replicase, (ATP,CTP):tRNA nucleotidyl-transferase and yeast tyrosyl-tRNA synthetase. The new fold has biological implications that can be and as a predictive tool for elaborating new experiments.