EVIDENCE FROM ULTRAVIOLET ABSORBANCE MEASUREMENTS FOR A CODON-INDUCED CONFORMATIONAL CHANGE IN LYSINE TRANSFER-RNA FROM ESCHERICHIA-COLI
EVIDENCE FROM ULTRAVIOLET ABSORBANCE MEASUREMENTS FOR A CODON-INDUCED CONFORMATIONAL CHANGE IN LYSINE TRANSFER-RNA FROM ESCHERICHIA-COLI
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DOI：
10.1073/pnas.76.7.3266
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发表时间：
1979-01-01
影响因子：
11.1
通讯作者：
GASSEN, HG
中科院分区：
文献类型：
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作者：
MOLLER, A;WILD, U;GASSEN, HG
从平衡透析的实验中，早先得出结论，密码子-反密码子复合物的形成触发了大肠杆菌tRNAPhe三级结构的构象变化。这里在聚（A）/tRNALys系统中证明了类似的构象转变。以C-G-A或C-G-A-A作为tRNA构象转换的探针。这些探针结合到tRNAPhe和tRNALys更强烈地在相应的密码子的存在下比在没有。为了通过独立的方法验证这些数据，使用在tRNALys（其含有2-硫代-5-甲基氨基甲基尿苷，s2 mam 5 U）中密码子-反密码子复合物形成时发生的300 nm处吸光度的降低。A3形成的结合常数。tRNALys（Ka = 2.4 . 10（4）M-1）和A4。tRNALys（Ka = 2.5 . 10（5）M-1）非常接近平衡透析获得的那些。在C-G-A存在下，A3与tRNA的表观结合常数提高10倍至2.5。10（-5）M-1。计算了C-G-A与二元配合物A3的结合常数。tRNALys约为2。10（4）M-1，而与游离tRNA的结合低于10（3）M-1。在适当的条件下，A3与tRNALys的结合可以通过加入C-G-A直接诱导。这些数据表明，密码子-反密码子复合物的形成诱导tRNA的构象变化，其结果是允许三核苷二磷酸的结合，推测为T-psi-G区域。
From experiments with equilibrium dialysis it was concluded earlier that formation of the codon-anticodon complex triggers a conformational change in the tertiary structure of tRNAPhe from Escherichia coli. A similar conformational transition is demonstrated here in the poly(A)/tRNALys system. C-G-A or C-G-A-A was used as a probe for the conformational transition in tRNA. These probes bound to tRNAPhe and tRNALys more strongly in the presence of the corresponding codons than in the absence. In order to verify these data by an independent method, the decrease in absorbance at 300 nm that occurs on formation of the codon-anticodon complex in tRNALys (which contains 2-thio-5-methylaminomethyluridine, s2mam5U) was used. The binding constants for formation of A3 . tRNALys (Ka = 2.4 . 10(4) M-1) and A4 . tRNALys (Ka = 2.5 . 10(5) M-1) are very close to those obtained by equilibrium dialysis. In the presence of C-G-A the apparent binding constant of A3 to tRNA was raised 10-fold to 2.5 . 10(-5) M-1. It was calculated that the constant for the binding of C-G-A to the binary complex A3 . tRNALys is approximately 2 . 10(4) M-1, whereas binding to the free tRNA is lower than 10(3) M-1. Under appropriate conditions binding of A3 to tRNALys can be induced directly by the addition of C-G-A. These data demonstrate that codon-anticodon complex formation induces a conformational change in the tRNA that as a consequence allows the binding of a trinucleoside diphosphate, presumably to the T-psi-G region.