Effect of synonymous mutations and codon context on mRNA translation

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CHEVANCE, Fabienne Françoise Valérie, 2017. Effect of synonymous mutations and codon context on mRNA translation [Dissertation]. Konstanz: University of Konstanz

@phdthesis{Chevance2017Effec-41437, title={Effect of synonymous mutations and codon context on mRNA translation}, year={2017}, author={Chevance, Fabienne Françoise Valérie}, address={Konstanz}, school={Universität Konstanz} }

<rdf:RDF xmlns:dcterms="http://purl.org/dc/terms/" xmlns:dc="http://purl.org/dc/elements/1.1/" xmlns:rdf="http://www.w3.org/1999/02/22-rdf-syntax-ns#" xmlns:bibo="http://purl.org/ontology/bibo/" xmlns:dspace="http://digital-repositories.org/ontologies/dspace/0.1.0#" xmlns:foaf="http://xmlns.com/foaf/0.1/" xmlns:void="http://rdfs.org/ns/void#" xmlns:xsd="http://www.w3.org/2001/XMLSchema#" > <rdf:Description rdf:about="https://kops.uni-konstanz.de/rdf/resource/123456789/41437"> <dspace:isPartOfCollection rdf:resource="https://kops.uni-konstanz.de/rdf/resource/123456789/28"/> <dcterms:isPartOf rdf:resource="https://kops.uni-konstanz.de/rdf/resource/123456789/28"/> <dc:contributor>Chevance, Fabienne Françoise Valérie</dc:contributor> <dspace:hasBitstream rdf:resource="https://kops.uni-konstanz.de/bitstream/123456789/41437/3/Chevance_2-1lx1wwrii79cj4.pdf"/> <dc:creator>Chevance, Fabienne Françoise Valérie</dc:creator> <foaf:homepage rdf:resource="http://localhost:8080/jspui"/> <dc:rights>terms-of-use</dc:rights> <dcterms:title>Effect of synonymous mutations and codon context on mRNA translation</dcterms:title> <dc:language>eng</dc:language> <dc:date rdf:datatype="http://www.w3.org/2001/XMLSchema#dateTime">2018-02-19T13:27:03Z</dc:date> <bibo:uri rdf:resource="https://kops.uni-konstanz.de/handle/123456789/41437"/> <void:sparqlEndpoint rdf:resource="http://localhost/fuseki/dspace/sparql"/> <dcterms:hasPart rdf:resource="https://kops.uni-konstanz.de/bitstream/123456789/41437/3/Chevance_2-1lx1wwrii79cj4.pdf"/> <dcterms:rights rdf:resource="https://kops.uni-konstanz.de/page/termsofuse"/> <dcterms:available rdf:datatype="http://www.w3.org/2001/XMLSchema#dateTime">2018-02-19T13:27:03Z</dcterms:available> <dcterms:abstract xml:lang="eng">Our interest on the effect of codon context on translation began in the lab with the isolation of a tRNA mutant (serT) that was defective in translation of the UCA codon for amino acid 7 of the regulatory flagellar protein FlgM (Chevance, 2006). There are dozens of UCA codons in the more than 50 genes required for flagellum production and chemotaxis. Therefore it was quite surprising that this serT mutant did not have an effect on motility. Why would the mutant apparently translate the many UCA codons of all the flagellar genes normally, but fail to efficiently translate the UCA codon in flgM? It seemed possible that the defect in flgM translation might be caused by context effects of codons flanking the UCA codon for amino acid 7 (Ser7) of FlgM. In order to monitor context effects on translational kinetics in a more general manner and under in vivo conditions, we developed a bacterial genetic system based on translation of the his operon leader peptide gene to determine the relative speed at which the ribosome reads single or multiple codons in vivo. Assaying translation through codon pairs for the 5’- and 3’- side positioning of the 64 codons relative to a specific codon revealed that the codon-pair orientation significantly affected in vivo translation speed. Codon pairs with rare arginine codons and successive proline codons were among the slowest codon pairs translated in vivo. This system allowed us to determine the effects of different factors on in vivo translation speed including the Shine-Dalgarno sequence, rate of peptide bond formation, codon context, and charged tRNA levels. The results of this study are reported in Chapter 1. We decided to revisit the codon context in the flagellar regulatory protein FlgM and mutated the codons adjacent to the UCA codon, with and without the serT-tRNA mutation. The effects on translation of replacing codons Thr6 and Pro8 of flgM with synonymous alternates produced a 600-fold range in FlgM activity. Synonymous changes at Thr6 and Leu9 resulted in a two fold range in FlgM activity. The level of FlgM activity produced by any codon arrangement was directly proportional to the degree of in vivo ribosome stalling at synonymous codons. Synonymous codon suppressors that corrected the effect of a translation-defective synonymous flgM allele were restricted to two codons flanking the translation-defective codon. The various codon arrangements had no apparent effects on flgM mRNA stability or predicted mRNA secondary structures. Our data suggest that efficient mRNA translation is determined by a triplet-of-triplet genetic code. That is, the efficiency of translating a particular codon is influenced by the nature of the immediately adjacent flanking codons. The results of this study are reported in Chapter 2. We also investigated codon context in the flagellar regulatory protein FliA and developed an assay to study context effects in the flagellin protein FliC. In the case of FliA, there was still an effect of codon context, but in this case, fliA mRNA stability was also affected. The stability of the flgM mRNA transcript during translation stalling was due to a translational restart at the end of flgM coding sequence. This translational restart protection of mRNA did not occur with fliA and prevented the distinction between the effects of codon context on translation versus mRNA stability for the fliA transcript. These observations are discussed in Chapter 3. These studies need to be extended to the case of other proteins and in other organisms in order to support whether the triplet-of-triplet genetic code is universal and further studies need to elucidate the mechanisms involved.</dcterms:abstract> <dcterms:issued>2017</dcterms:issued> </rdf:Description> </rdf:RDF>

Dateiabrufe seit 19.02.2018 (Informationen über die Zugriffsstatistik)

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