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Title: Identification and characterization of new messenger RNA interferases of Escherichia coli
Author: Christensen-Dalsgaard, Mikkel
ISNI:       0000 0004 2719 4706
Awarding Body: University of Newcastle Upon Tyne
Current Institution: University of Newcastle upon Tyne
Date of Award: 2011
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Toxin-antitoxin (TA) loci, which are widely distributed in prokaryotes, are organized in small operons, consisting of genes encoding toxins and antitoxins. Transcription from the promoter is regulated by the TA protein complex. Activation of the toxin occurs when the toxin is in excess of the antitoxin, for example during nutritional stress when the labile antitoxins are rapidly degraded by cellular proteases. The biological function of TA systems is debated and several different models have been proposed. Several of the TA loci encode messenger RNA interferases (mls) that inhibit translation by cleaving mRNA. Two types are known: those that cleave mRNA codons at the ribosomal A-site and those that cleave any RNA site-specifically. In this work, it was shown that the ml, YoeB cleaved mRNA strictly dependent on translation in vivo. Furthermore, it was shown that site-specific mRNA cleavage by MazF occurs independently of translation but that translation can seriously influence MazF cleavage efficiency. Expression of the toxin Doc of phage P1 was shown to mediate mRNA cleavage, though activation of the endogenous E. coli RelE ml. In a different study, three new ml encoding TA loci of E. coli were identified and characterized. Ectopic expression of the three ml genes lead to a rapid growth arrest caused by inhibition the global rate-of-translation. Furthermore, ml induced degradation of several different model RNAs were observed in vivo. Two of the mls cleaved only translated RNAs, similar to RelE and YoeB, whereas one ml cleaved both in coding and non-coding regions of the RNAs, resembling the properties of MazF and ChpBK toxins. Transcription of the three TA mRNAs was induced by various stressful conditions and dependent on the proteases Lon and Clp. In a different section of this work, a new synthetic lethal screen was developed using antisense RNA regulated R1 plasmids. The screen was used to search for synthetic lethal of sick interactions with the molecule responsible for trans-translation, tmRNA. Two genes were found to be synthetic sick with tmRNA: the tatC gene, which encodes an essential component of the twin- ariginine translocation complex and the dksA gene, which encode a transcription factor responsible for regulation of ribosomal RNA promoters.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available