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The emergence of the ribosome constituted a pivotal step in the evolution of life. This event happened nearly four billion years ago, and any traces of early stages of ribosome evolution are generally thought to have completely eroded away. Surprisingly, a detailed analysis of the structure of the modern ribosome reveals a concerted and modular scheme of its early evolution.

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The -1 programmed ribosomal frameshifts (PRF), which are used by many viruses, occur at a heptanucleotide slippery sequence and are currently thought to involve the tRNAs interacting with the ribosomal P- and A-site codons. We investigated here whether the tRNA occupying the ribosomal E site that precedes a slippery site influences -1 PRF. Using the human immunodeficiency virus type 1 (HIV-1) frameshift region, we found that mutating the E-site codon altered the -1 PRF efficiency. When the HIV-1 slippery sequence was replaced with other viral slippery sequences, mutating the E-site codon also altered the -1 PRF efficiency. Because HIV-1 -1 PRF can be recapitulated in bacteria, we used a bacterial ribosome system to select, by random mutagenesis, 16S ribosomal RNA (rRNA) mutations that modify the expression of a reporter requiring HIV-1 -1 PRF. Three mutants were isolated, which are located in helices 21 and 22 of 16S rRNA, a region involved in translocation and E-site tRNA binding. We propose a novel model where -1 PRF is triggered by an incomplete translocation and depends not only on the tRNAs interacting with the P- and A-site codons, but also on the tRNA occupying the E site.

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In the yeast Saccharomyces cerevisiae, nuclear DNA-encoded tRNA Lys CUU is partially imported into mitochondria. We previously found that the synthetic transcripts of yeast tRNA Lys and a number of their mutant versions could be specifically internalized by isolated yeast and human mitochondria. The mitochondrial targeting of tRNA Lys in yeast was shown to depend on the cytosolic precursor of mitochondrial lysyl-tRNA synthetase and the glycolytic enzyme enolase. Here we applied the approach of in vitro selection (SELEX) to broaden the spectrum of importable tRNA-derived molecules. We found that RNAs selected for their import into isolated yeast mitochondria have lost the potential to acquire a classical tRNA-shape. Analysis of conformational rearrangements in the importable RNAs by in-gel fluorescence resonance energy transfer (FRET) approach permitted us to suggest that protein factor binding and subsequent import require formation of an alternative structure, different from a classic L-form tRNA model. We show that in the complex with targeting protein factor, enolase 2, tRK1 adopts a particular conformation characterized by bringing together the 39-end and the TCC loop. This is a first evidence for implication of RNA secondary structure rearrangement in the mechanism of mitochondrial import selectivity. Based on these data, a set of small RNA molecules with significantly improved efficiency of import into yeast and human mitochondria was constructed, opening the possibility of creating a new mitochondrial vector system able to target therapeutic oligoribonucleotides into deficient human mitochondria.

A new RNA structural motif consisting of two double helices closely packed via minor grooves is found in many places in the ribosome structure. The packing requires that a GU base pair in one helix be packed against a Watson-Crick pair in the other helix. Two such motifs mediate the interaction of the P- and E-tRNA with the large ribosomal subunit. Analysis of the particular positions of these two motifs in view of the available data on occupancy of tRNA-binding sites and structural changes in the ribosome during the elongation cycle suggests a distinct role for each motif in tRNA translocation.