Wednesday, July 2, 2008

Modifications in ribosomal RNA

This, my Cand. Scient. (MS) project, was completed at the Department of Biological Chemistry, Institute of Molecular Biology, University of Copenhagen. My supervisor was Associate Professor Birte Vester who was part of the RNA group led by Professor Roger Garrett. Here I did three years of lab-work investigating posttranscriptional modifications of ribosomal RNA using standard lab methods combined with mass spectrometry. I was able to isolate the part of the 23S ribosomal RNA that constitutes the peptidyl transferase centre using site-directed RNaseH digestion followed by isolation with PAGE. The isolated fragments were analysed by MALDI-MS, where I was so fortunate to collaborate with Associate Professor Finn Kirpekar (University of Southern Denmark) who analyzed the fragments - and taught me to perform this type of analyzis on equipment in Copenhagen. I also screened these fragments for the mass-silent pseudouridines using chemical modification and detection with RT-PCR and visualization on sequencing gels. At the same time the first high resolution crystal structure of the ribosome was published, and I was able to use this data to visualize the spatial position of my modifications from five different bacteria and archaea, and showing that they were all positioned in the peptidyl transferase pocket facing outwards, away from the ribosomal RNA, and towards the amino-acetylated tRNAs. These findings suggest that the modifications may be important to the ribosome function, however, the positions of the modifications varied between species and some modifications were non-ubiquitous - so the precise function and importance remains unknown today.

During these years I learned that if you can work with RNA you can work with everything. And especially highly structured RNA like ribosomal RNA has a tendency of wanting to ball up in a tight structure expelling whatever probes you are trying to hybridize - no matter if these probes are of DNA or RNA or spiked with modified nucletotides such as LNA. The solution for me was to move probes to regions of less stability by trial and error. Also, I was introduced to Linux and have been running Debian ever since. This Linux platform was optimal for the visualization work I did using VMD. One image can be seen below, where the ribosome structure is shown in crown and side view with methylations shown in blue and psedouridines in red.

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