non-coding RNAs; Ribosome; Translation regulation; nucleotide analog interference
Gebetsberger Jennifer, Fricker Roger, Polacek Norbert (2015), cDNA library generation for the analysis of small RNAs by high-throughput sequencing., in Methods in Molecular Biology
Amort Melanie, Nachbauer Birgit, Tuzlak Selma, Kieser Arnd, Schepers Aloys, Villunger Andreas, Polacek Norbert (2015), Expression of the vault RNA protects cells from undergoing apoptosis., in Nature communications
, 6, 7030-7030.
Koch Miriam, Flür Sara, Kreutz Christoph, Ennifar Eric, Micura Ronald, Polacek Norbert (2015), Role of a ribosomal RNA phosphate oxygen during the EF-G-triggered GTP hydrolysis., in Proceedings of the National Academy of Sciences of the United States of America
, 112(20), 2561-8.
Koch Miriam, Clementi Nina, Rusca Nicola, Vögele Paul, Erlacher Matthias, Polacek Norbert (2015), The integrity of the G2421-C2395 base pair in the ribosomal E-site is crucial for protein synthesis., in RNA biology
, 12(1), 70-81.
Pircher Andreas, Bakowska-Zywicka Kamilla, Schneider Lukas, Zywicki Marek, Polacek Norbert (2014), An mRNA-derived noncoding RNA targets and regulates the ribosome., in Molecular Cell
Pircher Andreas, Gebetsberger Jennifer, Polacek Norbert (2014), Ribosome-associated ncRNAs: an emerging class of translation regulators., in RNA Biology
Gebetsberger Jennifer, Polacek Norbert (2013), Slicing tRNAs to boost functional ncRNA diversity., in RNA Biology
Molecular insight into ribosome functions and translation regulation1.SUMMARYThe ribosome is the largest known RNA enzyme and is regarded as one of the most ancient catalysts in biology. The ribosome, a multifunctional RNA-protein complex, translates the genome’s message into all proteins needed for life. Its crucial role in gene expression is mirrored by the fact that the ribosome represents the main target for clinical relevant antibiotics. Therefore the knowledge of its functioning is crucial for our understanding of antibiotic resistance and for the future design of new anti-microbial compounds. In this proposal we want to address two fundamental questions: (i)How does the ribosome function at the molecular and atomic level?(ii)How is the ribosome regulated during protein biosynthesis?To gain insight into these topics we will apply an experimental tool that has been established recently in my laboratory that allows manipulating individual functional groups or even single atoms of the large subunit ribosomal RNA. This technique significantly expands the chemical repertoire of residues that can be placed at specific sites within the ribosome and thus clearly exceeds the potential of standard mutagenesis. Here we want to investigate the role of two functional centers of the large ribosomal subunit, namely the ribosomal E-site as well as the peptidyl transferase center. Both of these sites have been shown to be targeted by antibiotics thus highlight their biological relevance. By establishing a novel in vitro selection regime we further want to ‘retro-evolve’ the large ribosomal subunit RNA in order to gain insight into the evolutionary history of this ancient RNA enzyme. Pivotal for cellular life is a well-controlled and coordinated gene expression system. Given the central position of translation in gene expression it is not surprising that protein synthesis is amply targeted by regulatory molecules, mostly regulatory proteins. More recently also small non-protein-coding RNAs (ncRNAs) have been added to the list of regulatory entities (e.g. microRNAs, anti-sense RNAs). However these ncRNAs regulating translation almost exclusively target the messenger RNAs rather than the ribosome directly. In this proposal we want to test the hypothesis, that small ncRNA do exist that directly bind to the ribosome and are capable of fine-tuning its activity. A recently started genomic screen in my group for small ncRNA ribo-regulators in the yeast Saccharomyces cerevisiae revealed hundreds of potential ribosome-associated ncRNAs. With the proposed research we now want to functionally characterize some of the most promising candidates to reveal their regulatory potential.Cumulatively, the proposed research will likely expand our current knowledge of ribosome functions and more precisely will shed new light on the catalytic and regulatory repertoire of non-protein-coding RNAs in biology.