Nazarenko I A, Uhlenbeck O C
Department of Chemistry and Biochemistry, University of Colorado at Boulder 80309-0215.
Biochemistry. 1995 Feb 28;34(8):2545-52. doi: 10.1021/bi00008a019.
A nuclease protection assay was used to obtain equilibrium dissociation constants of Thermus thermophilus EF-Tu with two well-characterized internal deletions of Escherichia coli Ala-tRNA(Ala) and yeast Phe-tRNA(Phe). Aminoacylated tRNAs with the anticodon hairpin substituted by a tetranucleotide bind to EF-Tu as well as the corresponding full-sized tRNAs. However, the Ala minihelix, where residue A7 is joined directly to A49, binds to EF-Tu less well than the full-sized Ala-tRNA(Ala). Similar data were obtained for Escherichia coli EF-Tu. An in vitro selection strategy was used to isolate a substrate for EF-Tu from an RNA library where nine random nucleotides inserted between A7 and A49 in the Ala minihelix. After six rounds of enrichment, two groups of RNA were obtained that bound T. thermophilus EF-Tu as well as Ala-tRNA(Ala). Group I molecules have the consensus sequence UNDUGACUY (N = U, C, A, G; D = U, G; Y = U, C) in the randomized region, and Group II molecules generally have 5'-terminal GUG, but are more variable in the remaining six nucleotides. The selected RNAs bind EF-Tu better than the minihelix either because they provide additional function groups for protein binding or because they have a structure more similar to the aminoacyl acceptor branch of tRNA.
采用核酸酶保护分析方法，获得嗜热栖热菌EF-Tu与两种特征明确的内部缺失型大肠杆菌丙氨酰tRNA（Ala）和酵母苯丙氨酰tRNA（Phe）的平衡解离常数。反密码子发夹被四核苷酸取代的氨酰化tRNA与EF-Tu的结合情况与相应的全长tRNA相同。然而，A7残基直接与A49相连的丙氨酸小螺旋与EF-Tu的结合能力不如全长丙氨酰tRNA（Ala）。对大肠杆菌EF-Tu也获得了类似的数据。采用体外筛选策略从一个RNA文库中分离出EF-Tu的底物，该文库中在丙氨酸小螺旋的A7和A49之间插入了9个随机核苷酸。经过六轮富集后，获得了两组与嗜热栖热菌EF-Tu以及丙氨酰tRNA（Ala）结合的RNA。第一组分子在随机区域具有共有序列UNDUGACUY（N = U、C、A、G；D = U、G；Y = U、C），第二组分子通常具有5'-末端GUG，但其余六个核苷酸的变异性更大。所选RNA与EF-Tu的结合能力优于小螺旋，这可能是因为它们为蛋白质结合提供了额外的功能基团，或者是因为它们具有与tRNA的氨酰受体臂更相似的结构。
