Yarus M, Cline S W, Wier P, Breeden L, Thompson R C
J Mol Biol. 1986 Nov 20;192(2):235-55. doi: 10.1016/0022-2836(86)90362-1.
In previous publications, we have shown that it is practical to study the translational activity of tRNAs by replacement and alteration of the anticodon arm sequence of the genus on a plasmid clone. Experiments in which the anticodon arm sequence is transplanted between tRNA genes suggest that the translational activity is determined by these sequences. We have therefore made every variant of the anticodon loop and the three base-pairs of the stem proximal to the loop, in order to resolve the relation between the structure of Su7Am tRNATrp, and its function. All derivatives conserved the normal secondary structure of the molecule, which was known to be essential for translational activity. The probability of translation of the amber codon by these suppressors is measured in this work. This translational activity in vivo is rationalized in terms of data on the copy numbers of the plasmid clones, the nucleotide modifications of the tRNAs, the steady-state level of the mature tRNA, and the aminoacylation of these molecules. Nucleotide modification levels vary among these tRNAs, giving information about the specificities of modification systems that make O-methylribose, pseudouridine, and modified A in the anticodon arm. However, for this series of tRNAs, none of these modifications has a strong effect on translational efficiency of the tRNAs. A few of the substitutions reduce aminoacylation of the tRNAs with glutamine, as determined by comparison of suppression in normal strains and related strains, which have 25-fold elevated levels of the glutaminyl-tRNA synthetase (GlnRS). The substitutions that have the largest effect on GlnRS action are, unexpectedly, purines for conserved pyrimidines on the 5' side of the anticodon loop. Data on the concentrations of tRNA in vivo suggest that the anticodon loop and helix contribute similarly to the determination of the steady-state level of the tRNAs. This level varies sevenfold, though all tRNAs are processed from a homologous precursor made from the same transcription unit. Effects on levels appear to be mediated by changes in anticodon arm structure. A robust equation that relates aminoacyl-tRNA levels to suppressor efficiency is developed in order to resolve effects on tRNA levels and on ribosomal steps: E = A/(K + A), where E is efficiency, A is aminoacyl-tRNA concentration, and K is the effective concentration, or cellular tRNA content required for an individual tRNA to have an efficiency of 0.50. The tRNAs vary in their intrinsic ability to function on the ribosome (represented by K), after other influences have been normalized.(ABSTRACT TRUNCATED AT 400 WORDS)
在之前的出版物中，我们已经表明，通过在质粒克隆上替换和改变该属的反密码子臂序列来研究tRNA的翻译活性是可行的。将反密码子臂序列在tRNA基因之间进行移植的实验表明，翻译活性由这些序列决定。因此，我们制备了反密码子环及其近端茎的三个碱基对的每一种变体，以解析Su7Am tRNATrp的结构与其功能之间的关系。所有衍生物都保留了分子的正常二级结构，已知该结构对翻译活性至关重要。在这项工作中测量了这些抑制子对琥珀密码子的翻译概率。体内的这种翻译活性根据质粒克隆的拷贝数、tRNA的核苷酸修饰、成熟tRNA的稳态水平以及这些分子的氨酰化数据进行了合理化分析。这些tRNA的核苷酸修饰水平各不相同，提供了有关在反密码子臂中产生O-甲基核糖、假尿苷和修饰A的修饰系统特异性的信息。然而，对于这一系列tRNA，这些修饰均未对tRNA的翻译效率产生强烈影响。通过比较正常菌株和谷氨酰胺-tRNA合成酶（GlnRS）水平升高25倍的相关菌株中的抑制情况，确定了一些取代会降低tRNA与谷氨酰胺的氨酰化作用。对GlnRS作用影响最大的取代出乎意料地是反密码子环5'侧保守嘧啶被嘌呤取代。体内tRNA浓度的数据表明，反密码子环和螺旋对tRNA稳态水平的决定作用相似。尽管所有tRNA均由来自同一转录单元的同源前体加工而来，但该水平变化了七倍。对水平的影响似乎是由反密码子臂结构的变化介导的。为了解析对tRNA水平和核糖体步骤的影响，建立了一个将氨酰-tRNA水平与抑制效率相关联的稳健方程：E = A/(K + A)，其中E是效率，A是氨酰-tRNA浓度，K是有效浓度，即单个tRNA效率达到0.50所需的细胞tRNA含量。在其他影响因素被归一化后，这些tRNA在核糖体上发挥功能的内在能力（由K表示）各不相同。（摘要截断于400字）
