Abstract
While all studies of protein synthesis to date have employed monoaminoacylated transfer RNAs, there have been reports that bisphenylalanyl-tRNA is formed by Thermus thermophilus phenylalanyl-tRNA synthetase. Such tandemly activated tRNAs have now been prepared by chemicoenzymatic techniques and are shown to function in both prokaryotic and mammalian protein synthesizing systems. They exhibit characteristics consistent with their possible utility under extreme conditions in natural systems and have important potential advantages for protein elaboration in cell free systems. Mechanistically, the bisaminoacylated tRNAs bind to the ribosomal A-site and utilize the aminoacyl moiety attached to the 3'-position of the terminal adenosine for addition to the growing polypeptide chain. Following translocation to the P-site and transfer of the formed peptidyl moiety, the donor tRNA dissociates from the ribosome as a monoaminoacylated tRNA capable of functioning in a subsequent polypeptide elongation step.
Highlights
In Vitro Protein Translation Reactions—Suppression of the UAG stop codon at position 27 of DHFR by mono- and bisaminoacyl-tRNACUAs was carried out using a rabbit reticulocyte lysate system (8), while suppression at positions Ϫ1 of DHFR and position 284 of firefly luciferase was performed in an E. coli S30 system (9)
We employed a general method for the preparation of bisaminoacylated tRNAs
This involved a modification of our published strategy for “chemical aminoacylation” (15, 16) in which an aminoacyl-pdCpA derivative was ligated to a tRNA lacking the 3Ј-terminal CpA
Summary
While all studies of protein synthesis to date have employed monoaminoacylated transfer RNAs, there have been reports that bisphenylalanyltRNA is formed by Thermus thermophilus phenylalanyl-tRNA synthetase. Such tandemly activated tRNAs have been prepared by chemicoenzymatic techniques and are shown to function in both prokaryotic and mammalian protein synthesizing systems. They exhibit characteristics consistent with their possible utility under extreme conditions in natural systems and have important potential advantages for protein elaboration in cell free systems. Following translocation to the P-site and transfer of the formed peptidyl moiety, the donor tRNA dissociates from the ribosome as a monoaminoacylated tRNA capable of functioning in a subsequent polypeptide elongation step
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