Reconstitution of the 50S Subunit with In Vitro-Transcribed 23S rRNA: a New Tool for Studying Peptidyltransferase
Philipp Khaitovich
Center for Pharmaceutical Biotechnology-m / c 870, University of Illinois, 900 S. Ashland Ave., Chicago, IL, 60607
Search for more papers by this authorAlexander S. Mankin
Center for Pharmaceutical Biotechnology-m / c 870, University of Illinois, 900 S. Ashland Ave., Chicago, IL, 60607
Search for more papers by this authorPhilipp Khaitovich
Center for Pharmaceutical Biotechnology-m / c 870, University of Illinois, 900 S. Ashland Ave., Chicago, IL, 60607
Search for more papers by this authorAlexander S. Mankin
Center for Pharmaceutical Biotechnology-m / c 870, University of Illinois, 900 S. Ashland Ave., Chicago, IL, 60607
Search for more papers by this authorRoger A. Garrett
Search for more papers by this authorSummary
RNA plays a critical role in all the steps of protein synthesis. The idea that the modern ribosome evolved from the original RNA-only protein-synthesizing machinery was so appealing, and made so much sense from the evolutionary standpoint, that it immediately found many proponents. The high degree of rRNA sequence conservation is usually perceived as an indication of the functional importance of the corresponding rRNA segment. Several nucleotide sequence segments of 23S rRNA, as well as a number of individual nucleotides, are invariant among all the studied organisms. Peptidyltransferase substrates, aminoacyl- and peptidyl-tRNAs, form tight contacts with rRNA, which has been demonstrated most clearly by crosslinking and footprinting experiments. By contrast, the omission of 5S rRNA during reconstitution of Thermus aquaticus 50S subunits with either natural or in vitro-transcribed 23S rRNA had a significantly more severe effect on the peptidyltransferase activity, which was reduced to the level of no more than 0.03% of that of native 50S subunits. The RNA component of the peptidyltransferase center is composed of RNA segments scattered in the 23S rRNA primary structure. While some rRNA sequences are brought together in the rRNA secondary structure, other elements are brought into proximity with the catalytic center in the tertiary structure of the 50S subunit.
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