Reliable semi-synthesis of hydrolysis-resistant 3′-peptidyl-tRNA conjugates containing genuine tRNA modifications

Abstract

The 3′-peptidyl-tRNA conjugates that possess a hydrolysis-resistant ribose-3′-amide linkage instead of the natural ester linkage would represent valuable substrates for ribosomal studies. Up to date, access to these derivatives is severely limited. Here, we present a novel approach for the reliable synthesis of non-hydrolyzable 3′-peptidyl-tRNAs that contain all the respective genuine nucleoside modifications. In short, the approach is based on tRNAs from natural sources that are site-specifically cleaved within the TΨC loop by using DNA enzymes to obtain defined tRNA 5′-fragments carrying the modifications. After dephosphorylation of the 2′,3′-cyclophosphate moieties from these fragments, they are ligated to the respective 3′-peptidylamino-tRNA termini that were prepared following the lines of a recently reported solid-phase synthesis. By this novel concept, non-hydrolyzable 3′-peptidyl-tRNA conjugates possessing all natural nucleoside modifications are accessible in highly efficient manner.

Binding of Macrolide Antibiotics Leads to Ribosomal Selection against Specific Substrates Based on Their Charge and Size

“…In order to substantiate this conclusion, we followed the kinetics of peptide bond formation between the donor MRL-tRNA and different model acceptor substrates. For these experiments, we synthesized chemically stable substrates mimicking the aminoacyl-tRNA acceptor end (Graber et al, 2010; Moroder et al, 2009) (Table 1). In these aminoacyl-tRNA analogs with the general structure ACCA-X, various amino acid residues (X) were linked to the 3′ carbon atom of the universal tRNA sequence ACCA via a stable amide bond (Supplementary Information).…”

The Structure of FemXWv in Complex with a Peptidyl‐RNA Conjugate: Mechanism of Aminoacyl Transfer from Ala‐tRNAAla to Peptidoglycan Precursors

“…Co-crystallization with RNA has been unsuccessful, which is presumably due to the low affinity of FemX Wv for free tRNA Ala , [4] instability of the Ala-tRNA Ala ester bond, and ordered fixation of the substrates, [5] that is, the peptidoglycan precursor followed by Ala-tRNA Ala , which hinders formation of a binary Fem/Ala-tRNA Ala complex. As duplexes containing less than five base pairs are unstable in solution, we connected RNA strands by a hexaethylene glycol linker [6,7] (Scheme 1 B). These compounds are composed of a peptidoglycan precursor analogue covalently linked to RNA moieties mimicking the acceptor arm of tRNA Ala .…”

The Synthesis of Methylated, Phosphorylated, and Phosphonated 3′‐Aminoacyl‐tRNASec Mimics

“…To generate full‐length tRNA Sec mimics that comprise the modified 3′‐termini, we elaborated an efficient enzymatic ligation strategy. A previously introduced concept for ligative tRNA synthesis utilizes 3′‐fragments that are 18 nt long and 5′‐fragments with a size of 58 nt 11. 31 The ligation site was positioned within the TΨC loop to allow proper annealing of the binary pre‐ligation complex required for T4 RNA ligase.…”