Oligonucleotide-directed peptide synthesis in a ribosome- and ribozyme-free system

Peptide bond formation by the ribosome requires 23S rRNA and its interactio n with the 3'-CCA end of tRNA. To investigate the possible evolutionary dev elopment of the peptidyl transfer reaction, we tried to obtain peptide bond formation without the ribosome or rRNA simply by using a piece of tRNA-an aminoacyl-minihelix-mixed with sequence-specific oligonucleotides that cont ained puromycin, Peptide bond formation was detected by gel electrophoresis , TLC analysis, and mass spectrometry. Peptide synthesis depended on sequen ce complementarity between the 3'-CCA sequence of the minihelix and the pur omycin-bearing oligonucleotide, However, proximity of the reacting species was not by itself sufficient for peptide bond formation. In addition, imida zole as a catalyst was required. Its role may be similar to the recently pr oposed mechanism, wherein A2451 of 235 rRNA works as a general base. Thus, peptide bond formation can be achieved with a simple, minimized system that captures the essence of an interaction seen in the ribosome.