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Ribozymes pp 215–226Cite as

Probing Functions of the Ribosomal Peptidyl Transferase Center by Nucleotide Analog Interference

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 848))

Abstract

The ribosome is a huge ribonucleoprotein complex in charge of protein synthesis in every living cell. The catalytic center of this dynamic molecular machine is entirely built up of 23S ribosomal RNA and therefore the ribosome can be referred to as the largest natural ribozyme known so far. The in vitro reconstitution approach of large ribosomal subunits described herein allows nucleotide analog interference studies to be performed. The approach is based on the site-specific introduction of nonnatural nucleotide analogs into the peptidyl transferase center, the active site located on the interface side of the large ribosomal subunit. This method combined with standard tests of ribosomal functions broadens the biochemical repertoire to investigate the mechanism of diverse aspects of translation considerably and adds another layer of molecular information on top of structural and mutational studies of the ribosome.

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Acknowledgments

We thank Ronald Micura and his team for a fruitful collaboration and for continuously providing top-quality synthetic RNA oligos. Prabhavathi Fernandes from Cempra Pharmaceuticals (Chapel Hill, NC, USA) is acknowledged for providing solithromycin. Nina Clementi and Anna Chirkova are thanked for experimental advice and comments on the manuscript. In addition we are grateful for constant support from Alexander Mankin, Knud Nierhaus, Wolfgang Piendl, and Alexander Hüttenhofer. Work in our laboratory is funded by grants from the Austrian Science Foundation FWF (Y315 to N.P. and P22658-B12 to M. E.) and the Austrian Ministry of Science and Research (GenAU project consortium “non-coding RNAs” D-110420-012-012 to N.P).

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Authors and Affiliations

  1. Division of Genomics and RNomics, Innsbruck Biocenter, Medical University of Innsbruck, Innsbruck, Austria

    Matthias D. Erlacher

  2. Department of Chemistry and Biochemistry, University of Bern, Bern, Switzerland

    Norbert Polacek

Authors
  1. Matthias D. Erlacher
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  2. Norbert Polacek
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Corresponding author

Correspondence to Norbert Polacek .

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Editors and Affiliations

  1. Department of Chemistry, Universität Konstanz, Universitätsstr. 10, Konstanz, 78457, Germany

    Jörg S. Hartig

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Erlacher, M.D., Polacek, N. (2012). Probing Functions of the Ribosomal Peptidyl Transferase Center by Nucleotide Analog Interference. In: Hartig, J. (eds) Ribozymes. Methods in Molecular Biology, vol 848. Humana Press. https://doi.org/10.1007/978-1-61779-545-9_14

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  • DOI: https://doi.org/10.1007/978-1-61779-545-9_14

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  • Publisher Name: Humana Press

  • Print ISBN: 978-1-61779-544-2

  • Online ISBN: 978-1-61779-545-9

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