Puglisi E V, Puglisi J D, Williamson J R, RajBhandary U L
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
Proc Natl Acad Sci U S A. 1994 Nov 22;91(24):11467-71. doi: 10.1073/pnas.91.24.11467.
An important step in initiation of protein synthesis in Escherichia coli is the specific formylation of the initiator methionyl-tRNA (Met-tRNA) by Met-tRNA transformylase. The determinants for formylation are clustered mostly in the acceptor stem of the initiator tRNA. Here we use NMR spectroscopy to characterize the conformation of two RNA microhelices, which correspond to the acceptor stem of mutants of E. coli initiator tRNA and which differ only at the position corresponding to the "discriminator base" in tRNAs. One of the mutant tRNAs is an extremely poor substrate for Met-tRNA transformylase, whereas the other one is a much better substrate. We show that one microhelix forms a structure in which its 3'-ACCA sequence extends the stacking of the acceptor stem. The other microhelix forms a structure in which its 3'-UCCA sequence folds back such that the 3'-terminal A22 is in close proximity to G1. These results highlight the importance of the discriminator base in determining tRNA conformation at the 3' end. They also suggest a correlation between tRNA structure at the 3' end and its recognition by Met-tRNA transformylase.
在大肠杆菌中，蛋白质合成起始的一个重要步骤是起始甲硫氨酰 - tRNA（Met - tRNA）被Met - tRNA转甲酰酶特异性甲酰化。甲酰化的决定因素大多聚集在起始tRNA的受体茎区。在这里，我们使用核磁共振光谱来表征两个RNA微螺旋的构象，它们对应于大肠杆菌起始tRNA突变体的受体茎区，并且仅在与tRNA中“判别碱基”相对应的位置有所不同。其中一个突变tRNA是Met - tRNA转甲酰酶的极差底物，而另一个则是更好的底物。我们表明，一个微螺旋形成一种结构，其中其3'-ACCA序列延伸了受体茎的堆积。另一个微螺旋形成一种结构，其中其3'-UCCA序列向后折叠，使得3'-末端的A22与G1紧密相邻。这些结果突出了判别碱基在确定tRNA 3'端构象中的重要性。它们还表明tRNA 3'端结构与其被Met - tRNA转甲酰酶识别之间存在相关性。
