Wurmbach P, Nierhaus K H
Proc Natl Acad Sci U S A. 1979 May;76(5):2143-7. doi: 10.1073/pnas.76.5.2143.
A method for binding tRNA to ribosomes, introduced by Watanabe [Watanabe, S. (1972) J. Mol. Biol. 67, 443-457], permits nonenzymatic binding of N-acetyl-Phe-tRNA(Phe) to either the ribosomal aminoacyl-tRNA (A) or peptidyl-tRNA (P) site with almost 100% specificity. We used this method to analyze a possible codon-anticodon interaction at the P site for NH(2)-blocked aminoacyl-tRNA and deacylated tRNA. N-Acetyl-Phe-tRNA(Phe) bound only to the P site of poly(U)-programmed 70S ribosomes, not to poly(A)-programmed ribosomes. The reverse mRNA dependence was found for N-acetyl-Lys-tRNA(Lys). A series of purified deacylated tRNAs was analyzed in the poly(U) and poly(A) system for abilities to block P-site binding of N-acetyl-aminoacyl-tRNA and to direct the N-acetyl-aminoacyl-tRNA to the A site. Only the cognate tRNA was as effective as the bulk tRNA at a concentration of less than 1/20th that of bulk tRNA. tRNAs whose corresponding codons are identical or similar (same base character) in the first two codon positions showed a low but significant effect. The other noncognate tRNAs were unable to direct the NH(2)-blocked aminoacyl-tRNAs to the A site. Chlortetracycline interfered neither with the P-site binding of NH(2)-blocked aminoacyl-tRNA nor with the effects of deacylated tRNAs. Furthermore, the translocation blocker viomycin affected neither the binding to the A site nor that to the P site. These effects of both antibiotics indicate that both kinds of tRNA do not bind transiently in the A site before filling the P site and that codon-anticodon interaction takes place at the P site.
渡边[渡边，S.（1972年）《分子生物学杂志》67卷，443 - 457页]介绍的一种使tRNA与核糖体结合的方法，能使N - 乙酰 - 苯丙氨酰 - tRNA（Phe）以几乎100%的特异性非酶促结合到核糖体氨酰 - tRNA（A）或肽酰 - tRNA（P）位点。我们用此方法分析了NH₂封闭的氨酰 - tRNA和脱酰基tRNA在P位点可能存在的密码子 - 反密码子相互作用。N - 乙酰 - 苯丙氨酰 - tRNA（Phe）仅结合到聚（U）编程的70S核糖体的P位点，不结合到聚（A）编程的核糖体。对于N - 乙酰 - 赖氨酰 - tRNA（Lys），发现了相反的mRNA依赖性。在聚（U）和聚（A）系统中分析了一系列纯化的脱酰基tRNA阻断N - 乙酰 - 氨酰 - tRNA与P位点结合以及将N - 乙酰 - 氨酰 - tRNA引导至A位点的能力。只有同源tRNA在浓度低于总tRNA浓度的1/20时，与总tRNA的效果相同。其相应密码子在前两个密码子位置相同或相似（相同碱基特征）的tRNA显示出较低但显著的效果。其他非同源tRNA无法将NH₂封闭的氨酰 - tRNA引导至A位点。金霉素既不干扰NH₂封闭的氨酰 - tRNA与P位点的结合，也不影响脱酰基tRNA的作用。此外，转位阻断剂紫霉素既不影响与A位点的结合，也不影响与P位点的结合。这两种抗生素的这些作用表明，两种tRNA在填充P位点之前都不会在A位点短暂结合，并且密码子 - 反密码子相互作用发生在P位点。
