Tetracycline regulates transcription and translation of tetO by stabilizing alternative folds of the leader RNA
Year: 2014
Type: dissertation
Abstract: Antibiotics belonging to the tetracycline family are characterized by their exceptional efficacy against many Gram-positive and Gram-negative bacteria, atypical bacteria such as rickettsia, chlamydia and mycoplasma and some protozoan parasites. Tetracyclines inhibit bacterial protein synthesis by binding to the 30S ribosomal subunit, thereby preventing stable accommodation of aminoacylated-tRNA at the ribosomal A-site. The extensive use has strongly limited their efficacy as antimicrobials due to the concomitant emergence and spread of microbial resistance. The most widely studied resistance mechanisms include active export of the antibiotic by efflux proteins and ribosomal protection proteins. The ribosomal protection protein TetO mediates resistance to tetracycline by promoting release of the antibiotic from the ribosome. But, since TetO and the elongation factor EF G bind to the same site on the ribosome, a high cellular concentration of TetO interferes with translation and is, thus, toxic for the cells. Hence, TetO expression needs to be tightly regulated. It already has been shown that the expression of tetO is induced by tetracycline and regulated by the 5´-untranslated region (5´-UTR) located directly upstream of the tetO start codon at the level of transcription and translation. Based on mutagenic studies and exhaustive RNA structure analysis, the different leader RNA conformations allowing tetracycline-mediated regulation of tetO expression were identified and characterized. This made it possible to establish a model according to which the expression of tetO is regulated by the formation of three RNA-structures. In the absence of tetracycline, an intrinsic transcription terminator prohibits the expression of tetO. Any non-terminated, read-through transcript adopts a conformation preventing subsequent translation of tetO. In it, the tetO ribosomal initiation site is occluded in a stem-structure stabilized by a pseudoknot interaction. The formation of these two leader RNA structures efficiently suppresses TetO synthesis in the absence of tetracycline. Only the presence of tetracycline stabilizes formation of an alternative RNA structure allowing both transcription and translation of the tetO open reading frame. In addition to the regions, which, due to their alternative sequence complementarity, provide the basis for leader RNA mediated regulation of tetO expression, a short upstream open reading frame (uORF) is also located in the 5´-region of the tetO leader sequence. The presence of this leader peptide sequence had already been shown to be required for tetracycline-mediated induction of tetO expression. However, the data obtained in this study strongly suggest that tetracycline induces tetO expression by directly contacting the nascent leader transcript and not just by stalling the ribosome at the uORF. These findings suggest a novel combined regulatory mechanism involving a ribosome as sensor and transmitter of tetracycline to the tetO leader RNA to initiate the expression of tetO via a tetracycline-dependent riboswitch. The structure elements required for tetracycline-mediated regulation of tetO expression are highly conserved among the 5´-UTRs of other ribosomal protection protein determinants. Thus, it seems reasonable to assume that their expression is accomplished by a common regulatory mechanism.
Author: Corina Heidrich
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W3211322957