Ricker R D, Kaji A
University of Pennsylvania, School of Medicine, Department of Microbiology, Philadelphia 19104-6076.
Nucleic Acids Res. 1991 Dec 11;19(23):6573-8. doi: 10.1093/nar/19.23.6573.
Single-stranded DNA (ssDNA) oligomers were compared to synthetic RNA oligomers in their ability to program E. coli ribosomes in vitro. AUG and dATG-containing oligomers promoted the non-enzymatic binding of fmet-tRNA to ribosomes, with similar dependence on time and magnesium concentration; only at 10 mM Mg++ or at low oligomer concentration was RNA slightly preferred in complex formation. These initiation complexes were biologically active in that fmet-tRNA, bound in response to ssDNA or RNA, was fully reactive with puromycin. While dAUG could not function as an initiation codon, p-dAUG functioned as well as AUG or dATG. However, dUAA and p-dUAA could not replace UAA in directing release-factor (RF) activity, and dTAA functioned only to a slight extent. Release factors had specificity for termination complexes containing dATGTAA, dATGTAG, or dATGTGA. At Mg++ concentrations of 15 mM or higher, these hexamers directed peptidyl transferase-dependent fmet-tRNA hydrolysis in the absence of RF. We suggest this RF-independent activation of peptidyl transferase as a unique system for studying the mechanism of termination. Overall, these results indicate that ssDNA can be used in place of RNA for certain studies of protein synthesis.
在体外对大肠杆菌核糖体进行编程的能力方面，将单链DNA（ssDNA）寡聚体与合成RNA寡聚体进行了比较。含AUG和dATG的寡聚体促进了甲硫氨酸起始tRNA（fmet-tRNA）与核糖体的非酶促结合，对时间和镁浓度的依赖性相似；只有在10 mM Mg++或低寡聚体浓度下，RNA在复合物形成中才略占优势。这些起始复合物具有生物学活性，因为响应ssDNA或RNA结合的fmet-tRNA与嘌呤霉素完全反应。虽然dAUG不能作为起始密码子，但对氨基苯甲酰-dAUG（p-dAUG）的功能与AUG或dATG一样好。然而，dUAA和p-dUAA在指导释放因子（RF）活性方面不能取代UAA，而dTAA仅在一定程度上起作用。释放因子对含有dATGTAA、dATGTAG或dATGTGA的终止复合物具有特异性。在Mg++浓度为15 mM或更高时，这些六聚体在没有RF的情况下指导肽基转移酶依赖性的fmet-tRNA水解。我们认为这种肽基转移酶的RF非依赖性激活是研究终止机制的独特系统。总体而言，这些结果表明，在某些蛋白质合成研究中，ssDNA可用于替代RNA。
