Anticodon sequence mutants of Escherichia coli initiator tRNA: effects of overproduction of aminoacyl-tRNA synthetases, methionyl-tRNA formyltransferase, and initiation factor 2 on activity in initiation.
Anticodon sequence mutants of Escherichia coli initiator tRNA: effects of overproduction of aminoacyl-tRNA synthetases, methionyl-tRNA formyltransferase, and initiation factor 2 on activity in initiation.
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DOI：
10.1021/bi034011r
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发表时间：
2003-05
期刊：
影响因子：
2.9
通讯作者：
C. Mayer;C. Köhrer;Eimear Kenny;Carsten Prusko;U. RajBhandary
中科院分区：
文献类型：
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作者：
C. Mayer;C. Köhrer;Eimear Kenny;Carsten Prusko;U. RajBhandary
大肠杆菌起始子tRNA的反密码子序列突变体用AUG以外的密码子和甲硫氨酸以外的氨基酸起始蛋白质合成。因为在许多情况下，反密码子序列对于氨酰-tRNA合成酶识别tRNA是重要的，所以突变的tRNA在体内被不同的氨基酸氨酰化。突变体tRNA在体内的起始活性取决于（i）tRNA的表达水平，（ii）tRNA的氨酰化程度，（iii）氨酰-tRNA转化为甲酰氨酰-tRNA（fAA-tRNA）的程度，以及（iv）fAA-tRNA对起始因子IF 2和核糖体的亲和力。以前，使用E.大肠杆菌过量生产氨酰-tRNA合成酶，甲硫氨酰-tRNA甲酰转移酶，或IF 2，我们确定了限制活性的步骤，在启动突变体tRNA氨酰化谷氨酰胺和缬氨酸。在这里，我们已经确定的步骤，限制活性的突变体tRNA的氨酰化与异亮氨酸和苯丙氨酸。涉及多种起始密码子（AUG、UAG、CAG、GUC、AUC和UUC）的实验的综合结果为核糖体.fAA-tRNA复合物可作为蛋白质合成起始的中间体的假设提供了支持。使用表面等离子体共振比较各种fAA-tRNA（fMet-、fGln-、fVal-、fIle-和fPhe-tRNA）与IF 2的结合亲和力支持IF 2可以充当fAA-tRNA与核糖体的载体的想法。其他结果表明，C1 xA 72碱基对错配，独特的真细菌和细胞器起始tRNA，也可能是重要的fAA-tRNA的结合IF 2。
Anticodon sequence mutants of Escherichia coli initiator tRNA initiate protein synthesis with codons other than AUG and amino acids other than methionine. Because the anticodon sequence is, in many cases, important for recognition of tRNAs by aminoacyl-tRNA synthetases, the mutant tRNAs are aminoacylated in vivo with different amino acids. The activity of a mutant tRNA in initiation in vivo depends on (i) the level of expression of the tRNA, (ii) the extent of aminoacylation of the tRNA, (iii) the extent of formylation of the aminoacyl-tRNA to formylaminoacyl-tRNA (fAA-tRNA), and (iv) the affinity of the fAA-tRNA for the initiation factor IF2 and the ribosome. Previously, using E. coli overproducing aminoacyl-tRNA synthetases, methionyl-tRNA formyltransferase, or IF2, we identified the steps limiting the activity in initiation of mutant tRNAs aminoacylated with glutamine and valine. Here, we have identified the steps limiting the activity of mutant tRNAs aminoacylated with isoleucine and phenylalanine. The combined results of experiments involving a variety of initiation codons (AUG, UAG, CAG, GUC, AUC, and UUC) provide support to the hypothesis that the ribosome.fAA-tRNA complex can act as an intermediate in initiation of protein synthesis. Comparison of binding affinities of various fAA-tRNAs (fMet-, fGln-, fVal-, fIle-, and fPhe-tRNAs) to IF2 using surface plasmon resonance supports the idea that IF2 can act as a carrier of fAA-tRNA to the ribosome. Other results suggest that the C1xA72 base pair mismatch, unique to eubacterial and organellar initiator tRNAs, may also be important for the binding of fAA-tRNA to IF2.