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Translation of specific small peptides on the ribosome can confer resistance to macrolide antibiotics. To reveal the molecular details of this and related phenomena, stable RNA-peptide conjugates that mimic peptidyl-tRNA would be desirable, especially for ribosome structural biology. A flexible solid-phase synthesis strategy now allows efficient access to these highly requested derivatives without restriction on the RNA and peptide sequences.

HIV-1 reverse transcriptase (RT) is a heterodimeric enzyme that converts the genomic viral RNA into proviral DNA. Despite intensive biochemical and structural studies, direct thermodynamic data regarding RT interactions with its substrates are still lacking. Here we addressed the mechanism of action of RT and of non-nucleoside RT inhibitors (NNRTIs) by isothermal titration calorimetry (ITC). Using a new incremental-ITC approach, a step-by-step thermodynamic dissection of the RT polymerization activity showed that most of the driving force for DNA synthesis is provided by initial dNTP binding. Surprisingly, thermodynamic and kinetic data led to a reinterpretation of the mechanism of inhibition of NNRTIs. Binding of NNRTIs to preformed RT/DNA complexes is hindered by a kinetic barrier and NNRTIs mostly interact with free RT. Once formed, RT/NNRTI complexes bind DNA either in a seemingly polymerase-competent orientation or form high-affinity dead-end complexes, both RT/NNRTI/DNA complexes being unable to bind the incoming nucleotide substrate.

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.

Flexibility exercised: Hydrolysis‐resistant 3′‐aminoacyl‐tRNA conjugates that contain a stable amide linkage instead of the natural ester are valuable substrates for biochemical studies of ribosomal processes. In a novel preparation of the stable E. coli initiator tRNA derivative 3′‐(N‐formylmethionyl)amino‐tRNAfMet the key feature is the synthesis of 3′‐azido oligoribonucleotides using a new functionalized solid support.

Graphical abstract

Die Translation kleiner Peptide am Ribosom kann diesem eine Resistenz gegen Macrolidantibiotika verleihen. Zur Aufklärung dieses und ähnlicher Phänomene sind stabile RNA‐Peptid‐Konjugate, die Peptidyl‐tRNA imitieren, wünschenswert, besonders für die strukturbiologische Untersuchung von Ribosomen. Eine flexible Festphasensynthesestrategie macht diese Konjugate nun in effizienter Weise zugänglich, ohne Beschränkungen bezüglich der RNA‐ und Peptidsequenz.