Hamann C S, Hou Y M
Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
Biochemistry. 1995 May 16;34(19):6527-32. doi: 10.1021/bi00019a034.
The discriminator base U73 at the acceptor terminus of Escherichia coli tRNA(Cys) is a determinant for the specific aminoacylation of this tRNA by the cognate cysteine tRNA synthetase. Substitution of U73 has a major deleterious effect on the catalytic efficiency of aminoacylation. Here, we show that an RNA hairpin minihelix and an RNA hairpin microhelix that recreate, respectively, the 12-base pair acceptor-T psi C stem and the 7-base pair acceptor helix of E. coli tRNA(Cys) were aminoacylated with cysteine. As in tRNA(Cys), alteration of U73 to A73, C73, or G73 in the cysteine mini- and microhelices eliminated aminoacylation. This established that the strong influence of U73 on aminoacylation is fully retained from the full-length tRNA(Cys) to the mini- and microhelixCys. Transfer of U73 to the noncognate minihelixAla conferred cysteine acceptance to the latter, despite the presence of the major determinant for alanine tRNA synthetase. Even minihelixGly, which shares U73 with minihelixCys, was an efficient substrate for aminoacylation with cysteine. Conversely, as long as U73 was present in minihelixCys, introduction of the glycine or alanine determinant could not block charging by cysteine tRNA synthetase. Although the catalytic efficiency of aminoacylation of these small RNA helices with cysteine was reduced by orders of magnitude from that of tRNA(Cys), the single nucleotide U73 determines the ability of these RNA helices to be aminoacylated with cysteine. These results demonstrated a dominant role of U73 for aminoacylation of small RNA helices by cysteine tRNA synthetase.
大肠杆菌tRNA（Cys）受体末端的鉴别碱基U73是该tRNA被同源半胱氨酸tRNA合成酶特异性氨酰化的决定因素。U73的取代对氨酰化的催化效率有重大有害影响。在此，我们表明，分别重现大肠杆菌tRNA（Cys）的12个碱基对受体-TψC茎和7个碱基对受体螺旋的RNA发夹小螺旋和RNA发夹微螺旋被半胱氨酸氨酰化。与tRNA（Cys）一样，半胱氨酸小螺旋和微螺旋中U73变为A73、C73或G73会消除氨酰化。这表明U73对氨酰化的强烈影响从全长tRNA（Cys）到小螺旋和微螺旋Cys都完全保留。将U73转移到非同源小螺旋Ala赋予后者半胱氨酸接受性，尽管存在丙氨酸tRNA合成酶的主要决定因素。甚至与小螺旋Cys共享U73的小螺旋Gly也是用半胱氨酸进行氨酰化的有效底物。相反，只要U73存在于小螺旋Cys中，引入甘氨酸或丙氨酸决定因素就不能阻止半胱氨酸tRNA合成酶的充电。尽管这些小RNA螺旋用半胱氨酸进行氨酰化的催化效率比tRNA（Cys）降低了几个数量级，但单核苷酸U73决定了这些RNA螺旋被半胱氨酸氨酰化的能力。这些结果证明了U73在半胱氨酸tRNA合成酶对小RNA螺旋氨酰化中的主导作用。
