tRNA hopping: effects of mutant tRNAs

School of Biological Sciences, University of Missouri-Kansas City, 5007 Rockhill Road, Kansas City, MO 64110, USA.
Biochimica et Biophysica Acta (Impact Factor: 4.66). 11/2003; 1630(1):41-6. DOI: 10.1016/j.bbaexp.2003.09.002
Source: PubMed


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.

2 Reads
  • [Show abstract] [Hide abstract]
    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 · 4.33 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Mutants of translation components which compensate for both -1 and +1 frameshift mutations showed the first evidence for framing malleability. Those compensatory mutants isolated in bacteria and yeast with altered tRNA or protein factors are reviewed here and are considered to primarily cause altered P-site realignment and not altered translocation. Though the first sequenced tRNA mutant which suppressed a +1 frameshift mutation had an extra base in its anticodon loop and led to a textbook "yardstick" model in which the number of anticodon bases determines codon size, this model has long been discounted, although not by all. Accordingly, the reviewed data suggest that reading frame maintenance and translocation are two distinct features of the ribosome. None of the -1 tRNA suppressors have anticodon loops with fewer than the standard seven nucleotides. Many of the tRNA mutants potentially affect tRNA bending and/or stability and can be used for functional assays, and one has the conserved C74 of the 3' CCA substituted. The effect of tRNA modification deficiencies on framing has been particularly informative. The properties of some mutants suggest the use of alternative tRNA anticodon loop stack conformations by individual tRNAs in one translation cycle. The mutant proteins range from defective release factors with delayed decoding of A-site stop codons facilitating P-site frameshifting to altered EF-Tu/EF1alpha to mutant ribosomal large- and small-subunit proteins L9 and S9. Their study is revealing how mRNA slippage is restrained except where it is programmed to occur and be utilized.
    Microbiology and molecular biology reviews: MMBR 04/2009; 73(1):178-210. DOI:10.1128/MMBR.00010-08 · 14.61 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    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
Show more