tRNA hopping: effects of mutant tRNAs.
ABSTRACT Movement of tRNA and mRNA through the ribosome is coupled. However, selection for suppression of a -1 frameshift mutation in Escherichia coli has yielded a class of mutant tRNAs that can violate this mechanism and "hop" or disengage from their cognate codons and re-pair downstream in the mRNA. Previously described tRNA mutants of this class included those with insertions in the anticodon of tRNA(Val)1. This report describes further tRNA(Val)1 alterations that enhance hopping; these include a novel insertion in the anticodon loop, base substitutions in the anticodon stem and a base deletion in the variable loop. These results indicate that several different features of a tRNA are important for maintaining stable codon-anticodon interactions and coupled movement of tRNA and mRNA during the elongation phase of protein synthesis.
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ABSTRACT: The hypothesis that tRNA sidearm loops bear anticodons assumes crossovers between anticodon and sidearms, or translation by expressed aminoacylated tRNA halves forming single stem-loops. Only the latter might require ribosomal adaptations. Drosophila mitochondrial codon usages coevolve with sidearm numbers bearing matching putative anticodons (comparing different codon families in one genome, macroevolution) and when comparing different genomes for single codon families (microevolution). Coevolution between Drosophila and yeast mitochondrial antisense tRNAs and codon usages partly confounds microevolutionary patterns for putative sidearm anticodons. Some tRNA sidearm loops have more than seven nucleotides, putative expanded anticodons potentially matching quadruplet codons (tetracodons, codons expanded by a fourth silent position, forming tetragenes (predicted by alignment analyses of Drosophila mitochondrial genomes)). Tetracodon numbers coevolve with expanded tRNA sidearm loops. Sidearm coevolution with amino acid usages and tetragenes occurs for putative anticodons in 5' and 3' sidearms loops (D and TΨC loops, respectively), are stronger for the D-loop. Results slightly favour isolated stem-loops upon crossover hypotheses. An alternative hypothesis, that patterns observed for sidearm 'anticodons' do not imply translational activity, but recognition signals for tRNA synthetases that aminoacylate tRNAs, is incompatible with tetracodon/tetra-anticodon coevolution. Hence analyses strengthen translational hypotheses for tRNA sidearm anticodons, tetragenes, and antisense tRNAs.Journal of Theoretical Biology 09/2013; DOI:10.1016/j.jtbi.2013.08.030 · 2.30 Impact Factor
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ABSTRACT: Introduction: We investigated frameshift suppressor tRNAs previously reported to use five-base anticodon-codon interactions in order to provide a collection of frameshift suppressor tRNAs to the synthetic biology community and to develop modular frameshift suppressor logic devices for use in synthetic biology applications. Results and Discussion: We adapted eleven previously described frameshift suppressor tRNAs to the BioBrick cloning format, and built three genetic logic circuits to detect frameshift suppression. The three circuits employed three different mechanisms: direct frameshift suppression of reporter gene mutations, frameshift suppression leading to positive feedback via quorum sensing, and enzymatic amplification of frameshift suppression signals. In the course of testing frameshift suppressor logic, we uncovered unexpected behavior in the frameshift suppressor tRNAs. The results led us to posit a four-base binding hypothesis for the frameshift suppressor tRNA interactions with mRNA as an alternative to the published five-base binding model. Conclusion and Prospects: The published five-base anticodon/codon rule explained only 17 of the 58 frameshift suppression experiments we conducted. Our deduced four-base binding rule successfully explained 56 out of our 58 frameshift suppression results. In the process of applying biological knowledge about frameshift suppressor tRNAs to the engineering application of frameshift suppressor logic, we discovered new biological knowledge. This knowledge leads to a redesign of the original engineering application and encourages new ones. Our study reinforces the concept that synthetic biology is often a winding path from science to engineering and back again; scientific investigations spark engineering applications, the implementation of which suggests new scientific investigations.Interdisciplinary Bio Central 09/2012; 4(10):1-12. DOI:10.4051/ibc.2012.4.3.0010
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ABSTRACT: If a ribosome shifts to an alternative reading frame during translation, the information in the message is usually lost. We have selected mutants of Salmonella typhimurium with alterations in tRNA(cmo5UGG)(Pro) that cause increased frameshifting when present in the ribosomal P-site. In 108 such mutants, two parts of the tRNA molecule are altered: the anticodon stem and the D-arm, including its tertiary interactions with the variable arm. Some of these alterations in tRNA(cmo5UGG)(Pro) are in close proximity to ribosomal components in the P-site. The crystal structure of the 30S subunit suggests that the C-terminal end of ribosomal protein S9 contacts nucleotides 32-34 of peptidyl-tRNA. We have isolated mutants with defects in the C-terminus of S9 that induce +1 frameshifting. Combinations of changes in tRNA(cmo5UGG)(Pro) and S9 suggest that an interaction occurs between position 32 of the peptidyl-tRNA and the C-terminal end of S9. Together, our results suggest that the cause of frameshifting is an aberrant interaction between the peptidyl-tRNA and the P-site environment. We suggest that the "ribosomal grip" of the peptidyl-tRNA is pivotal for maintaining the reading frame.Journal of Molecular Biology 12/2008; 385(2):350-67. DOI:10.1016/j.jmb.2008.10.069 · 3.96 Impact Factor