Molecular biology of ribonucleoprotein complexes

Members

• Melanie Amort M.Sc., PhD student
• Kamilla Bakowska-Zywicka Ph.D., postdoc
• Nina Clementi M.Sc., PhD student
• Matthias Erlacher Ph.D., postdoc
• Armin Fuchs, PhD student
• Jennifer Gebetsberger M.Sc., PhD student
• Birgit Nachbauer Master student
• Andreas Pircher, Master student
• Norbert Polacek Ph.D., Associate Prof.
• Krista Trappl M.Sc., Ph.D. student
• Marek Zywicki Ph.D., postdoc

Projects

- Molecular aspects of protein synthesis

- Probing ribosome functions by 23S rRNA nucleotide analogue interference

- Genomic screens for non-coding RNAs involved in translation regulation

project leader: Norbert Polacek, supported by the Austrian Science Foundation (FWF), (START award Y315 to N.P.; Lise-Meitner grant M1074 to K. B.-Z.), and the GenAU program (Project D-110420-012-012 to N. P.).

Ribosomes are multifunctional ribonucleoprotein (RNP) complexes that translate the genome's message into proteins needed for life in every living cell. As they are so fundamental to life and represent one of the main targets for antibiotics, comprehending how they work is at the heart of molecular understanding of biology. Decades of biochemical and recent crystallographic studies revealed the ribosome as an RNA-enzyme (ribozyme) with roots in the 'RNA world'. From an evolutionary point of view it is fascinating that the modern ribosome, the mother of all protein enzymes, still relies on RNA catalysis to synthesize proteins.
The aim of the project is to gain molecular insight into fundamental ribosomal reactions by employing a newly developed in vitro reconstitution assay of ribosomal particles. This allows the site-specific incorporation of a single modified nucleotide analog into 23S rRNA and to study the functional effects on the performance of the peptidyl transferase center. With this procedure, it is possible to identify crucial functional groups in the active site with improved precision and an enlarged chemical variety of nucleosides compared to regular mutagenesis. The main focus of our group lies in the elucidation of molecular aspects of peptide bond formation and peptide release; the two main reactions promoted by the peptidyl transferase center during the elongation and termination phases of protein synthesis, respectively. In addtion, we are studing the molecular requirements of elongation factor G (EF-G) driven tRNA translocation and ask how the large ribosomal subunit is assembled into a functionally competent RNP particle.

Project-relevant Publications

1. Polacek, N., Gaynor, M., Yassin, A. and Mankin, AS. (2001). Ribosomal peptidyl transferase can withstand mutations at the putative catalytic nucleotide. Nature 441:498-501.
2. Polacek, N., Gomez, MJ., Ito, K., Xiong, L., Nakamura, Y., and Mankin, AS. (2003). The critical role of the universally conserved A2602 of 23S ribosomal RNA in the release of the nascent peptide during translation termination. Mol Cell 11:103-112.
3. Erlacher, MD., Lang, K., Shankaran, N., Wotzel, B., Huettenhofer, A., Micura, R., Mankin, AS. and Polacek, N. (2005). Chemical engineering of the peptidyl transferase center reveals an important role of the 2-hydroxyl group of A2451. Nucleic Acids Res 33:1618-1627.
4. Erlacher, MD., Lang, K., Wotzel, B., Rieder, R., Micura, R., and Polacek, N. (2006). Efficient ribosomal peptidyl transfer critically relies on the presence of the ribose 2'-OH at A2451 of 23S rRNA. J Am Chem Soc 128:4453-4459.
5. Amort, M., Wotzel, B., Bakowska-Zywicka, K., Erlacher, MD., Micura, R., and Polacek, N. (2007). An intact ribose moiety at A2602 of 23S rRNA is key to trigger peptidyl-tRNA hydrolysis during translation termination. Nucleic Acids Res. 35:5130-5140.
6. Lang, K., Erlacher, MD., Wilson, DN., Micura, R., and Polacek, N. (2008). The Role of 23S Ribosomal RNA Residue A2451 in Peptide Bond Synthesis Revealed by Atomic Mutagenesis Chem Biol 15:485-492.
7. Clementi,N., Chirkova, A., Puffer, B., Micura, R., and Polacek, N. (2010). Atomic mutagenesis reveals A2660 of 23S ribosomal RNA as key to EF-G GTPase activation. Nature Chem. Biol. 6:344-351.
8. Chirkova, A., Erlacher, M. D., Clementi, N., Zywicki, M., Aigner, M., and Polacek, N. (2010). The role of the universally conserved A2450-C2063 base pair in the ribosomal peptidyl transferase center. Nucleic Acids Res. 38:4844-4855.

- Atomic mutagenesis of the small ribosomal subunit

project leader: Matthias Erlacher, supported by the Austrian Science Foundation (FWF), (P22658 to M. E.).

Project-relevant Publications

Wieland, M., Berschneider, B., Erlacher, M. D.,and Hartig, J. S.(2010). Aptazyme-mediated regulation of 16S ribosomal RNA. Chem Biol. 17:236-242.

- The elusive molecular biology of the vault RNA and the vault RNP

project leader: Norbert Polacek, supported by the Medizinische Forschungsförderung Innsbruck (MFI project D110420-012-011) and the Austrian Academy of Sciences (DOC-fFORTE stipend 23170 to M. Amort)

In a recent genomic non-coding RNA (ncRNA) screen we have identified the vault RNP associated RNAs to be significantly up-regulated in human B-cells upon Epstein-Barr virus (EBV) infection. Very little is known about the function of this ncRNA class, mainly because the vault complex has been overlooked by cell biologists for many years. Here we would like to address the question whether vault RNA up-regulation is indeed causally linked to the virus infection and which function the vault RNAs might possess during virus propagation. Furthermore we will assess the structure-function relationship of vault RNAs which will eventually reveal the so far enigmatic molecular biology of this interesting ncRNA species.

Project-relevant Publications

1. Hüttenhofer, A., Schattner, P. and Polacek, N. (2005). Non-coding RNAs: hope or hype? Trends in Genet. 21:289-297.
   
2. Mrazek, J., Kreutmayer, S. B., Grässer, F. A., Polacek, N., and Hüttenhofer, A.(2007). Subtractive hybridization identifies novel differentially expressed ncRNA species in EBV-infected human B cells. Nucleic Acids Res. 35: e73.
3. Nandy, C., Mrazek, J., Stoiber, H., Grässer, F.A., Hüttenhofer, A., and Polacek, N. (2009). Epstein-Barr virus-induced expression of a novel human vault RNA. J. Mol. Biol. 388:776-784.