Research: Ribosome structure and function

Ribosomes perform a universal and necessary biochemical process. 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 new techniques involving crosslinking and other physical techniques that allow us to determine intramolecular distances and distance changes in the ribosome. UV irradiation is an attractive method because native, highly active ribosomes can be used. As a complementary approach, photochemical reagents ( psoralens, psoralen derivatives and 4-thiouridine) are being targeted to specific sites in the 16S rRNA prior to reconstitution into 30S ribosomal subunits. These have been characterized for their functional activity and structure and usually are active and, thus, they can be used in the same experiments as native subunits. The 16S rRNA consists of 1542 nucleotides having a well studied secondary structure.

We have determined conformational changes in the 16S rRNA in, and around, the decoding region during ribosome subunit association and during association with initiation factor IF3. We speculate that these may be related to the movement of the tRNA during the elongation cycle and also consistent with other information that the 30S pre-initiation complex contains initiator tRNA bound in a stand-by site different than the P site. In a third experiment, we've detected specific tertiary structure changes upon the binding of some antibiotics known to interact with the 30S subunit and affect its activity. The successes in these experiments have provided new distance constraints that are useful in constructing and testing three dimensional models and also demonstrate that the structural approach we are taking will have the sensitivity needed to reveal higher order changes in the rRNA during translation.

Related links:

Ribosomal Database Project

Database of Ribosomal Crosslinks (DRC)