Tandemly Activated tRNAs as Participants in Protein Synthesis

Abstract

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 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.Aminoacyl-tRNAs are key intermediates in protein biosynthesis. These adaptor molecules (1) not only faithfully recognize the appropriate mRNA codon but also transfer aminoacyl residues onto the growing peptide chain, thus bridging the information gap between nucleic acids and proteins. The amino acid residue is attached to the 2Ј(3Ј)-hydroxyl group of the 3Ј-terminal adenosine residue of tRNA via an ester linkage. The esterification of a tRNA with an amino acid is mediated by an aminoacyl-tRNA synthetase. There are 20 different aminoacyl-tRNA synthetases, each having a cognate amino acid and substrate tRNA. While the attachment of the amino acid occurs specifically either on the 2Ј-or 3Ј-OH group of the 3Ј-terminal adenosine moiety in tRNA (2), the resulting aminoacyl ester is quite activated and equilibriates rapidly between the 2Ј-and 3Ј-positions (3-5).Stepanov et al. (6, 7) have described the formation of bis(2Ј,3Ј-O-phenylalanyl)-tRNAs by phenylalanyl-tRNA synthetase from Thermus thermophilus; the enzyme was shown to form a tandemly activated tRNA using T. thermophilus tRNA Phe as substrate, as well as Escherichia coli tRNA Phe and an RNA transcript identical in sequence with E. coli tRNA Phe . While the formation of bisphenylalanyl-tRNAs by T. thermophilus phenylalanyl-tRNA synthetase has been known for some time, possible functions of bisacylated tRNAs have apparently not been studied. This may reflect the difficulty in obtaining such species by conventional methods on a preparative scale.Presently, we have prepared several tRNAs having amino acids attached to both the 2Ј-and 3Ј-positions of the 3Ј-terminal adenosine moiety. The ability of these tandemly activated tRNAs to participate in protein synthesis is described, as is an analysis of the mechanistic details of that participation. EXPERIMENTAL PROCEDURESConstruction of Expression Plasmids-Plasmids pThis15 and pTHD8 were used to express wild-type DHFR 2 in different in vitro translation systems. Plasmid pThis15 was used in a rabbit reticulocyte lysate system (8),...

Improved amber and opal suppressor tRNAs for incorporation of unnatural amino acids in vivo. Part 1: Minimizing misacylation

“…These experiments show that both suppressor tRNAs are equally protected by EF-1a and/or other components of the translational machinery for at least 3.5 h, which has not been observed in vitro with or without EF-Tu (Nakata et al 2006;Wang et al 2006). Most importantly, this shows that aaRS editing and/or hydrolysis (Fig.…”

“…The tRNAamino acid bond is highly labile at physiological pH. In a recent study, a sample of dCA-Val at pH 7.8 without translational machinery components was 69% hydrolyzed after a 1-h incubation (Wang et al 2006). The cytoplasm of a Xenopus oocyte is estimated to be between pH 7.3 and 7.7 (Webb and Nuccitelli 1981;Sasaki et al 1992), and therefore some hydrolysis of tRNA-dCA-W would be expected after 3.5 h, if not protected by other proteins.…”

Elongation factor G stabilizes the hybrid-state conformation of the 70S ribosome

“…7E), in agreement with earlier findings (Belitsina et al 1975(Belitsina et al , 1979). An alternative interpretation of these results could be that NAc-Phe-tRNA Phe has the ability to bind in the hybrid P/E state, as there are findings to suggest that aminoacylated tRNA can bind to the 50S E site (Wang et al 2006). We Phe that is puromycin reactive: (pretrans) the pre-translocation complex as described in A; (GDPNP) non-hydrolyzable GTP analog; (fus) fusidic acid.…”

Evolution of translational machinery: Could translation termination come into being before elongation?

“…If the pH becomes acidic, hydrolysis rate would drop even more. It was shown recently, however, that tandemly acylated tRNAs bearing aminoacid residues both on 3 0 and 2 0 are much more stable than mono-acylated (Wang et al, 2006). These bis-acylated tRNAs are naturally used by translational apparatus of Thermus thermophilus (Stepanov et al, 1992(Stepanov et al, , 1998 and possibly contribute to the resistance of this organism to high temperatures.…”

“…At neutral pH at 37 1C it takes about 1 h to hydrolyse half of the aa-tRNA population (Wang et al, 2006). In comparison with rates of ordinary enzymatic reactions it is Eternity.…”