Ribosomes perform the fundamental biochemical process of protein synthesis in a highly conserved way in all organisms. The structure of both ribosomal subunits determined by x-ray crystallography structures indicate that the important active sites are composed of rRNA elements. It has been proposed for some time that conformational changes in the ribosome accompany many important steps in protein synthesis. However, this has been difficult to investigate because of the absence of techniques that can determine and monitor higher order structure in large RNAs. We have developed techniques to evaluate UV irradiation-induced RNA-RNA crosslinking that allow us to determine intramolecular distances and distance changes in the ribosome. As a complementary approach, photochemical reagents (psoralens, psoralen derivatives and 4-thiouridine) have been targeted to specific sites in the rRNA prior to reconstitution into 30S ribosomal subunits. These have been characterized for their functional activity and structure and they can be used in the same experiments as native subunits.
We have determined conformational changes in the 16S rRNA in, and around, the decoding region during ribosome subunit association, titration with Mg+2, during association with initiation factor IF3, and in response to tRNA binding to the P-site. We speculate that these structure changes are related to changes in the ribosome that occur during the movement of the tRNA during the elongation cycle. Experiments with initiator tRNA and IF3 are also consistent with information that the 30S pre-initiation complex contains initiator tRNA bound in a stand-by site different than the P site. In another experiment, we have detected specific tertiary structure changes upon the binding of some antibiotics known to interact with the 30S subunit. These experiments provided new distance constraints that are useful in constructing and testing three dimensional models and they also demonstrate that the approach we are taking has the sensitivity to reveal higher order changes in the rRNA during translation.
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Supplemental material for Huggins, W., Shapkina, T. and Wollenzien, P. 2007. Conformational energy and structure in canonical and non-canonical forms of tRNA determined by temperature analysis of the rate of s4U8-C13 photocrosslinking, RNA, in press. Link