Tandemly Activated tRNAs as Participants in Protein

phenylalanyl-tRNAsynthetase.Such tandemly activated tRNAs have now been prepared by chemicoen-zymatic techniques and are shown to function in both prokaryotic andmammalian protein synthesizing systems. They exhibit characteristicsconsistent with their possible utility under extreme conditions in naturalsystems and have important potential advantages for protein elaborationincellfreesystems.Mechanistically,thebisaminoacylatedtRNAsbindtothe ribosomal A-site and utilize the aminoacyl moiety attached to the3 -positionoftheterminaladenosineforadditiontothegrowingpolypep-tidechain.FollowingtranslocationtotheP-siteandtransferoftheformedpeptidyl moiety, the donor tRNA dissociates from the ribosome as amonoaminoacylated tRNA capable of functioning in a subsequentpolypeptide elongation step.Aminoacyl-tRNAs are key intermediates in protein biosynthesis. Theseadaptor molecules (1) not only faithfully recognize the appropriate mRNAcodon but also transfer aminoacyl residues onto the growing peptide chain,thus bridging the information gap between nucleic acids and proteins. Theaminoacidresidueisattachedtothe2 (3 )-hydroxylgroupofthe3 -terminaladenosine residue of tRNA via an ester linkage. The esterification of a tRNAwith an amino acid is mediated by an aminoacyl-tRNA synthetase. There are20 different aminoacyl-tRNA synthetases, each having a cognate amino acidand substrate tRNA. While the attachment of the amino acid occurs specifi-cally either on the 2 -or3 -OH group of the 3 -terminal adenosine moiety intRNA (2), the resulting aminoacyl ester is quite activated and equilibriatesrapidly between the 2 - and 3 -positions (3–5).Stepanov