Mechanism of a novel, complex riboswitch regulating expression of the ribosome protecting protein TetO

• Applicant: Dr. Christian Berens, since 10/2010
• Subject Area: Metabolism, Biochemistry and Genetics of Microorganisms
• Term: from 2008 to 2015
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 65226726

Expression of the ribosome protection protein TetO is inducible by tetracycline and autorepressed. Regulation of transcription requires a 273 bp leader sequence 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 postulate a novel, complex riboswitch triggered by simultaneous action of a ribosome and tetracycline. The resulting structural change yield leads to antitermination. We propose to identify the involved sequence elements by randomized and oligonucleotide directed mutagenesis. The structures of the leader RNA in the presence and absence of the ligands shall be determined by in vivo and in vitro probing. The binding sites of the ribosome and tetracycline shall be uncovered by interference analyses, using enzymatic and chemical RNA cleavage. The mechanism of autoregulation by the TetO protein will be studied using the same methods. We will determine the binding constants of tetracycline and TetO to the leader RNA, and to the RNA-ribosome complex, using fluorescence titration and surface plasmon resonance.These results should quantify the assumed ribosome-tetracycline interaction in the complex with RNA.

• DFG Programme: Research Grants
• Ehemaliger Antragsteller: Professor Dr. Wolfgang Hillen, until 10/2010 (†)