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A tRNA-mimic Strategy to Explore the Role of G34 of tRNA Gly in Translation and Codon Frameshifting

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A tRNA-mimic Strategy to Explore the Role of G34 of tRNA Gly in Translation and Codon Frameshifting

Aurélie Janvier et al. Int J Mol Sci.

Abstract

Decoding of the 61 sense codons of the genetic code requires a variable number of tRNAs that establish codon-anticodon interactions. Thanks to the wobble base pairing at the third codon position, less than 61 different tRNA isoacceptors are needed to decode the whole set of codons. On the tRNA, a subtle distribution of nucleoside modifications shapes the anticodon loop structure and participates to accurate decoding and reading frame maintenance. Interestingly, although the 61 anticodons should exist in tRNAs, a strict absence of some tRNAs decoders is found in several codon families. For instance, in Eukaryotes, G34-containing tRNAs translating 3-, 4- and 6-codon boxes are absent. This includes tRNA specific for Ala, Arg, Ile, Leu, Pro, Ser, Thr, and Val. tRNAGly is the only exception for which in the three kingdoms, a G34-containing tRNA exists to decode C3 and U3-ending codons. To understand why G34-tRNAGly exists, we analysed at the genome wide level the codon distribution in codon +1 relative to the four GGN Gly codons. When considering codon GGU, a bias was found towards an unusual high usage of codons starting with a G whatever the amino acid at +1 codon. It is expected that GGU codons are decoded by G34-containing tRNAGly, decoding also GGC codons. Translation studies revealed that the presence of a G at the first position of the downstream codon reduces the +1 frameshift by stabilizing the G34•U3 wobble interaction. This result partially explains why G34-containing tRNAGly exists in Eukaryotes whereas all the other G34-containing tRNAs for multiple codon boxes are absent.

Keywords: IRES element; frameshifting; genetic code; glycine codon; tRNA; translation.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure A1
Figure A1
Distribution of nucleotide +1 downstream of GGU Gly codons in each of the 23 eukaryotic genomes. 35,246,252 Gly codons were analysed. Results are expressed as percentages. The x-axis corresponds to the organisms while the y-axis refers to the occurrence of each nucleotide at position +1. Details of the analysis are shown below the graph.
Figure A2
Figure A2
The effect on translation of the Mg2+ concentration added to the extracts. R-Luc activity was measured in the 0 frame. Translation efficiencies are represented as raw bioluminescence activity (Relative Light Units or RLU) and resulted from at least 3 independent experiments. Standard deviations are shown.
Figure A3
Figure A3
Control of the integrity of the RNA constructs used in the study. RNA samples (0.4–0.6 μg) were fractionated by 4% denaturing PAGE and stained with ethidium bromide. (A) RNAs used in Figure 4; (B) RNAs used in Figure 5; (C) RNAs used in Figure 6; (D) RNAs used in Figure 7.
Figure A4
Figure A4
Analysis of the IGR-dependent translation products in vitro. For each construct, the 0- and +1-frame products were analysed. The reporter constructs were incubated in RRL translation extracts at 30 °C for 60 min in the presence of [35S]Met. Translation experiments were performed in untreated RRL containing endogenous lipoxygenase mRNA as well as globin mRNA in large amount (the globin band is not shown). Translation products were visualized by autoradiography after resolving on a 10% SDS PAGE.
Figure 1
Figure 1
Distribution of nucleotide 34 in tRNAs specific for 3, 4 and 6 codon boxes according to the amino acceptor identity. The bars represent the usage of each nucleotide 34 in the subgroups containing tRNAGly alone or tRNAs specific for Ala, Arg, Ile, Leu, Pro, Ser, Thr and Val. G34 is overrepresented in tRNAGly and nearly absent in tRNAs specific for Ala, Arg, Ile, Leu, Pro, Ser, Thr and Val.
Figure 2
Figure 2
Distribution of tRNAGly isoacceptors and Gly codon usage in a representative set of eukaryotes. (A) Histogram representing the distribution of tRNAGly isoacceptor genes per organism expressed as percentages (data from http://gtrnadb.ucsc.edu/). (B) The codon reading properties of tRNAsGly are represented with boxes using the colour code used in the histograms of panels A and C. The decoding properties of the rarely found tRNAGlyA34CC (modified in I34CC) are represented with a dashed green box. (C) The histogram represents the codon usage for Gly codons per organism, expressed as percentages (data from http://www.kazusa.or.jp/codon/).
Figure 3
Figure 3
Distribution of nucleotide +1 downstream from the four Gly codons in 23 eukaryotic genomes. 35,246,252 Gly codons were analysed. Results are expressed as percentages. The standard deviation bars are showing the dispersion between the genomes. The x-axis corresponds to the nucleotides found at position +1 after the four Gly codons.
Figure 4
Figure 4
Translation of R-Luc driven by the CrPV IGR and analysis of potential +1-frame translation. (A) The secondary structure of the IGR IRES. The IGR is driven translation of Renilla luciferase (R-Luc) fused in the 0-frame or +1-frame. To monitor the +1-frameshifting activity, one extra A nucleotide was added in the R-Luc sequence. Nucleotides and amino acid residues in blue are from R-Luc. The pseudoknot PKI which mimics the codon/anticodon is drawn in pink. The A-, P- and E-site of the ribosome are schematized. Nucleotide numbering is from the CrPV genome. (B) Histogram representing the luciferase activities in 0-frame or +1-frame starting from the WT or knockout PKI. Transcripts were synthesized in vitro, denatured/renatured and incubated in Rabbit Reticulocyte Lysates at 30 °C for 2 h. Translation of R-Luc was measured by measuring the bioluminescence produced with a Dual-Glo luciferase assay (Promega) in a luminometer. Translation efficiencies are represented as raw bioluminescence activity (Relative Light Units or RLU) and resulted from at least 3 independent experiments. Standard deviations are shown.
Figure 5
Figure 5
The effect on translation of the first nucleotide downstream the wild-type pseudo knot (PKI). (A) Histogram representing the luciferase activities of the synthesized R-Luc in 0-frame or +1-frame. Translation efficiencies are represented as raw bioluminescence activity (Relative Light Units or RLU) and resulted from at least 3 independent experiments. Standard deviations are shown. The construct with a G+1 corresponds to the WT PKI. (B) The same data represented as a 100% stack bar graph.
Figure 6
Figure 6
The impact on translation of the first nucleotide downstream of the PKI reprogrammed with a Gly GGU codon. (A) Histogram representing the luciferase activities of the synthesized R-Luc in 0-frame or +1-frame. Translation efficiencies are represented as raw bioluminescence activity (Relative Light Units or RLU) and resulted from at least 3 independent experiments. Standard deviations are shown. The first construct corresponds to the WT PKI. (B) The same data represented as a 100% stack bar graph.
Figure 7
Figure 7
The effect on translation of reprogramming the PKI with different Gly codon-anticodon combinations. (A) Schematic representations of the different RNAs tested. Non Watson-Crick base pairs are highlighted in yellow. (B) Histogram representing the luciferase activities of the synthesized R-Luc in 0-frame or +1-frame. Translation efficiencies are represented as raw bioluminescence activity (Relative Light Units or RLU) and resulted from at least 3 independent experiments. Standard deviations are shown. Construct names are described as XXX/YYY-G where X represent anticodon triplets, Y codon triplets, all with a G in +1 and written in the 5′-3′ direction. The first construct corresponds to the WT PKI.

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