Lustig F, Borén T, Claesson C, Simonsson C, Barciszewska M, Lagerkvist U
Department of Medical Biochemistry, University of Göteborg, Sweden.
Proc Natl Acad Sci U S A. 1993 Apr 15;90(8):3343-7. doi: 10.1073/pnas.90.8.3343.
We have investigated the influence of structures in the tRNA anticodon loop and stem on the ability of the anticodon to discriminate among codons. We had previously shown that anticodon UCC, when placed in the structural context of tRNA(Gly1) from Escherichia coli, discriminated efficiently between the glycine codons, as required by the wobble rules. Thus, this anticodon read GGA and GGG but did not read GGU and GGC, whereas in mycoplasma tRNA(Gly), the same anticodon did not discriminate among the glycine codons. We have now determined the reading properties of three constructions based on tRNA(Gly1) containing the anticodon UCC in different structural contexts. In one of these constructs, tRNA(Gly1-ASL), the anticodon loop and stem are the same as in mycoplasma tRNA(Gly). The second construct, tRNA(Gly1-AS), has an anticodon stem identical with the mycoplasma tRNA(Gly), whereas in the last construct, tRNA(Gly1-C32), the only difference from tRNA(Gly1)(UCC) is that the uridine in position 32 of the anticodon loop has been replaced by cytidine. These constructs were tested for ability to read glycine codons in an in vitro protein-synthesizing system that allowed us to monitor separately the reading of each codon. We found that the anticodon UCC, when present in tRNA(Gly1-AS), discriminated among the glycine codons, whereas in the constructs tRNA(Gly1-ASL) and tRNA(Gly1-C32), the same anticodon had lost its ability to discriminate--i.e., it behaved as in mycoplasma tRNA(Gly). These results strongly suggest that nt 32 of the anticodon loop of tRNA(Gly1)(UCC) decisively influences the reading properties of the anticodon UCC.
我们研究了tRNA反密码子环和茎的结构对反密码子区分密码子能力的影响。我们之前已经表明，当反密码子UCC处于来自大肠杆菌的tRNA(Gly1)的结构背景中时，它能按照摆动规则有效地区分甘氨酸密码子。因此，这个反密码子识别GGA和GGG，但不识别GGU和GGC，而在支原体tRNA(Gly)中，相同的反密码子不能区分甘氨酸密码子。我们现在确定了基于tRNA(Gly1)构建的三种结构在不同结构背景下含反密码子UCC的阅读特性。在其中一种构建体tRNA(Gly1-ASL)中，反密码子环和茎与支原体tRNA(Gly)中的相同。第二种构建体tRNA(Gly1-AS)具有与支原体tRNA(Gly)相同的反密码子茎，而在最后一种构建体tRNA(Gly1-C32)中，与tRNA(Gly1)(UCC)的唯一区别是反密码子环第32位的尿苷被胞苷取代。在一个体外蛋白质合成系统中测试了这些构建体读取甘氨酸密码子的能力，该系统使我们能够分别监测每个密码子的读取情况。我们发现，当反密码子UCC存在于tRNA(Gly1-AS)中时，它能区分甘氨酸密码子，而在构建体tRNA(Gly1-ASL)和tRNA(Gly1-C32)中，相同的反密码子失去了区分能力——即它的行为与支原体tRNA(Gly)中的一样。这些结果强烈表明，tRNA(Gly1)(UCC)反密码子环的第32位核苷酸决定性地影响反密码子UCC的阅读特性。
