Pagel F T, Murgola E J
Department of Molecular Genetics, University of Texas M.D., Anderson Cancer Center, Houston 77030, USA.
Gene Expr. 1996;6(2):101-12.
In 1984, our laboratory reported the characterization of the first misacylated tRNA missense suppressor, a mutant Escherichia coli lysine tRNA with a C70 to U base change in the amino acid acceptor stem. We suggested then that the suppressor tRNA, though still acylated to a large extent with lysine, is partially misacylated with alanine. The results reported in this article demonstrate that is the case both in vitro and in vivo. For the in vitro studies, the mutant tRNA species was isolated from the appropriate RPC-5 column fractions and shown to be acylatable with both lysine and alanine. For the in vivo demonstration, use was made of a temperature-sensitive alaS mutation, which results in decreasing acylation with Ala as the temperature is increased, resulting ultimately in lethality at 42 degrees C. The alaSts mutation was also used to demonstrate that the ability of the same missense suppressor, lysT(U70), to suppress a trpA frameshift mutation is not affected by the Ala-acylation deficiency. We conclude that the misacylation and altered decoding are two independent effects of the C70 to U mutation in tRNA(Lys). The influence of an alteration in the acceptor stem, which is in contact with the large (50S) ribosomal subunit, on decoding, which involves contact between the anticodon region of tRNA and the small (30S) ribosomal subunit, may occur intramolecularly, through the tRNA molecule. Alternatively, the U70 effect may be accomplished intermolecularly; for example, it may alter the interaction of tRNA with ribosomal RNA in the 50S subunit, which may then influence further interactions between the two subunits and between the 30S subunit and the anticodon region of the tRNA. Preliminary evidence suggesting some form of the latter explanation is presented. The influence of a single nucleotide on both tRNA identity and decoding may be related to the coevolution of tRNAs, aminoacyl-tRNA synthetases, and ribosomes.
1984年，我们实验室报道了首个错酰化tRNA错义抑制子的特性，它是一种突变的大肠杆菌赖氨酸tRNA，在氨基酸接受茎中存在C70到U的碱基变化。当时我们认为，这种抑制子tRNA虽然在很大程度上仍被赖氨酸酰化，但也有部分被丙氨酸错酰化。本文报道的结果表明，在体外和体内都是如此。对于体外研究，从合适的RPC - 5柱级分中分离出突变tRNA种类，并证明它可被赖氨酸和丙氨酸酰化。对于体内证明，利用了温度敏感型alaS突变，随着温度升高，该突变导致丙氨酰化减少，最终在42℃时导致致死性。alaSts突变还用于证明，同一个错义抑制子lysT(U70)抑制trpA移码突变的能力不受丙氨酰化缺陷的影响。我们得出结论，错酰化和译码改变是tRNA(Lys)中C70到U突变的两个独立效应。与大（50S）核糖体亚基接触的接受茎的改变对译码的影响，译码涉及tRNA的反密码子区域与小（30S）核糖体亚基之间的接触，可能通过tRNA分子在分子内发生。或者，U70效应可能通过分子间实现；例如，它可能改变tRNA与50S亚基中核糖体RNA的相互作用，这可能进而影响两个亚基之间以及30S亚基与tRNA反密码子区域之间的进一步相互作用。文中给出了一些初步证据，表明存在某种形式的后一种解释。单个核苷酸对tRNA识别和译码的影响可能与tRNA、氨酰 - tRNA合成酶和核糖体的共同进化有关。
