19F nuclear magnetic resonance as a probe of anticodon structure in 5-fluorouracil-substituted Escherichia coli transfer RNA.
19F nuclear magnetic resonance as a probe of anticodon structure in 5-fluorouracil-substituted Escherichia coli transfer RNA.
复制标题
19F 核磁共振作为 5-氟尿嘧啶取代的大肠杆菌转移 RNA 中反密码子结构的探针。
DOI：
10.1016/0022-2836(87)90565-1
复制
发表时间：
1987
影响因子：
5.6
通讯作者：
Horowitz,J
中科院分区：
文献类型：
--
作者：
Gollnick,P;Hardin,CC;Horowitz,J
使用19 F核磁共振（n.m.r.）通过研究四核苷酸与5-氟尿嘧啶取代的大肠杆菌RNA 1Val（反密码子FAC）.19Fn.m.r.结合的影响，扩展了光谱作为反密码子结构探针的应用范围。在不存在和存在不同浓度的具有序列GpUpApX（X = A，G，C，U）的寡核苷酸的情况下获得光谱，所述寡核苷酸含有缬氨酸密码子GpUpA。通过检测位于反密码子环33和34位的5-氟尿嘧啶残基，以及该分子的所有其他环和茎，来监测tRNA的结构变化。GpUpApA与反密码子和5′-相邻的FUra 33互补，结合后19 F光谱中有两个共振移动。一，峰值H（3.90 p. p.m.），也被GpUpA移位，并且先前在反密码子的摆动位置处被分配给FUra 34。GpUpApA的作用与GpUpA的作用不同之处在于，四核苷酸诱导第二共振峰F（4. 5 p. p.m.）的低场位移，在19 F标记的tRNA 1 Val的19 F光谱中。证据表明，含有密码子的寡核苷酸结合到反密码子的甲基质子光谱的6-甲基腺苷残基相邻的反密码子和从切割的tRNA在反密码子的RNase H结合后dGpTpApA，脱氧类似物的核糖核苷酸密码子。GpUpApA与氟化tRNA 1Val结合的结合常数，通过Scatchard分析n.m.r.结果，与其他方法得到的值吻合良好。在此基础上，我们将F峰归属于~（19）F核磁共振谱中。19 F标记tRNA 1 Valto FUra 33的光谱。在导致反密码子环裂解的条件下，用核酸酶S1部分水解后，氟化tRNA 1Valare的19 F光谱中的峰F和H的强度特异性降低，这一观察结果支持了峰H归属于FUra 34和先前峰H归属于FUra 34。只有当腺苷位于四核苷酸的3′-位时，峰F才发生低场位移; GpUpApG、GpUpApC或GpUpApU的结合只会导致峰H的高场位移。讨论了3′-末端腺苷和33位5-氟尿嘧啶残基之间的碱基特异性相互作用涉及反密码子环的5′-堆叠构象的可能性。证据还提出了温度依赖性的构象变化的反密码子环以下的tRNA的解链温度。
The use of19F nuclear magnetic resonance (n.m.r.) spectroscopy as a probe of anticodon structure has been extended by investigating the effects of tetranucleotide binding to 5-fluorouracil-substitutedEscherichia colitRNA1Val(anticodon FAC).19F n.m.r. spectra were obtained in the absence and presence of different concentrations of oligonucleotides having the sequence GpUpApX (X = A, G, C, U), which contain the valine codon GpUpA. Structural changes in the tRNA were monitoredviathe 5-fluorouracil residues located at positions 33 and 34 in the anticodon loop, as well as in all other loops and stems of the molecule. Binding of GpUpApA, which is complementary to the anticodon and the 5′-adjacent FUra 33, shifts two resonances in the19F spectrum. One, peak H (3.90 p.p.m.), is also shifted by GpUpA and was previously assigned to FUra 34 at the wobble position of the anticodon. The effects of GpUpApA differ from those of GpUpA in that the tetranucleotide induces the downfield shift of a second resonance, peak F (4.5 p.p.m.), in the19F spectrum of19F-labeled tRNA1Val. Evidence that the codon-containing oligonucleotides bind to the anticodon was obtained from shifts in the methyl proton spectrum of the 6-methyladenosine residue adjacent to the anticodon and from cleavage of the tRNA at the anticodon by RNase H after binding dGpTpApA, a deoxy analog of the ribonucleotide codon. The association constant for the binding of GpUpApA to fluorinated tRNA1Val, obtained by Scatchard analysis of the n.m.r. results, is in good agreement with values obtained by other methods. On the basis of these results, we assign peak F in the19F n.m.r. spectrum of19F-labeled tRNA1Valto FUra 33. This assignment and the previous assignment of peak H to FUra 34 are supported by the observation that the intensities of peaks F and H in the19F spectrum of fluorinated tRNA1Valare specifically decreased after partial hydrolysis with nuclease S1under conditions leading to cleavage in the anticodon loop. The downfield shift of peak F occurs only with adenosine in the 3′-position of the tetranucleotide; binding of GpUpApG, GpUpApC, or GpUpApU results only in the upfield shift of peak H. The possibility is discussed that this base-specific interaction between the 3′-terminal adenosine and the 5-fluorouracil residue at position 33 involves a 5′-stacked conformation of the anticodon loop. Evidence also is presented for a temperature-dependent conformational change in the anticodon loop below the melting temperature of the tRNA.