Poster abstracts

Poster number 48 submitted by Shijie Huang

Bacterial cell can live without free 5S rRNA

Shijie Huang (Center for Biomolecular Sciences, University of Illinois, Chicago), Dorota Klepacki (Center for Biomolecular Sciences, University of Illinois, Chicago), Luc Jaeger (University of California, Santa Barbara), Alexander S. Mankin (Center for Biomolecular Sciences, University of Illinois, Chicago)

Abstract:
The 120 nucleotide-long 5S rRNA is a universal component of cytoplasmic ribosomes of all living organisms. Together with the 23S rRNA and a handful of ribosomal proteins it assembles into the bacterial large ribosomal subunit. 5S rRNA has been suggested to be critical for ribosome assembly, to play a role in the catalysis of peptide bond formation, participate in signal transmission within the ribosome and be involved in other activities. However, even after decades of research, the true function of 5S rRNA remains unclear. The lack of new tools and methodologies hinders the progress towards the understanding the role of 5S rRNA in translation.
To develop a novel system for analyzing the functions of 5S rRNA in the living cell and to implement new approaches to enable ribosome engineering, we succeeded in fusing 5S with 23S rRNA into a single molecule. This was achieved by inserting circularly-permuted 5S rRNA at two different 23S rRNA locations. The resulting 23S-5S rRNA hybrid molecule assembles into a functional large ribosomal subunit. Furthermore, we were able to engineer E. coli cells in which all the ribosomes carry the 23S-5S hybrid rRNA and, therefore, we have created a cell which completely lacks free 5S rRNA. These results unequivocally show that free 5S rRNA is dispensable for the ribosome assembly or function.
We have also introduced 5S rRNA into Ribo-T, the ribosome with tethered small and large subunits, which has been previously built upon a hybrid 16S-23S rRNA molecule. The preliminary result shows resulting single-RNA ribosome is capable of catalyzing polypeptide formation. We envision that the 23S-5S hybrid or the single-RNA ribosome could be powerful new tools for studying 5S rRNA functions, biogenesis and evolution. The single-RNA ribosome makes it possible to create an orthogonal translation system in the cell which does not exchange any of its RNA components with the ‘housekeeping’ ribosome.

Keywords: Ribosome, 5S rRNA, Orthogonal translation