Mechanism of a novel, complex riboswitch regulating expression of the ribosome protecting protein TetO
Final Report Abstract
Tetracycline is a highly active, broad-spectrum antibiotic with only few negative side-effects that is easy and cheap to produce. Its consequent wide-spread usage has led to the emergence and dissemination of many resistance determinants. Those encoding ribosome protection proteins are the most frequently encountered in tetracycline-resistant bacteria. They encode a protein which binds to the ribosome and expels the bound antibiotic. Overexpression of ribosome protection proteins, like TetO, is toxic for the cell, because they out-compete elongation factors for binding to the ribosome. Therefore, their expression has to be tightly controlled to prevent deleterious effects on bacterial physiology. So far, how this regulation occurs is still mostly unknown. TetO expression is induced by tetracycline and autorepressed by TetO. Regulation requires a 273 bp 5’ untranslated region in the tetO mRNA containing a short open reading frame, a transcriptional terminator and a putative tetracycline binding site. Since all three elements are necessary for regulation, we postulated a novel, complex riboswitch triggered by simultaneous action of a ribosome and tetracycline. Reporter gene assays, Northern blots and quantitative PCR data showed that tetO expression is regulated both at the level of transcription and at the level of translation. A transcriptional terminator regulates the expression of tetO at the level of transcription. This terminator is the most stable secondary structure element in the entire 5’ untranslated region. It dominates folding of the corresponding in vitro transcribed RNA, preventing any tetracycline-dependent effects from being observed in chemical and enzymatic probing experiments. Even in the presence of tetracycline, about 90% of all transcripts are terminated. Therefore, all experiments regarding the “On-State” structures of the 5’ untranslated region had to be performed with mutants that were not able to form the terminator anymore. Translational stalling at the leader peptide caused by tetracycline binding to the ribosome is not the sole reason for transcription termination, because other antibiotics that induce stalling of the ribosome do not induce expression of tetO. Extensive mutagenesis studies and exhaustive RNA structure analysis led to a model proposing that translational regulation of tetO expression is controlled by the formation of two different RNA structures. In the absence of tetracycline, any non-terminated, read-through transcript adopts a conformation preventing subsequent translation of tetO by occluding the tetO ribosomal initiation site in a stem-structure stabilized by a pseudoknot interaction. Solely the presence of tetracycline stabilizes formation of an alternative RNA structure allowing both transcription and translation of the tetO open reading frame. The structure elements required for tetracycline-mediated regulation of tetO expression are highly conserved among the 5´-UTRs of many other ribosomal protection protein determinants. Thus, it is reasonable to assume that their expression is also controlled by a common regulatory mechanism.
Publications
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(2014) Probing RNA structure and ligand binding sites on RNA by Fenton cleavage. In: Handbook of RNA Biochemistry (E. Westhof, A. Bindereif, A. Schön, R. K. Hartmann, eds.). WILEY-VCH, Weinheim, Germany, Kap. 15
C.G. Heidrich, C. Berens
