Biochemical investigations of macromolecular orientations and interactions in the ribosome

We have used a combination of biochemical techniques to study macromolecular interactions and orientations in the ribosome. The interaction between 30S ribosomal proteins S6 and S18 was identified by yeast two-hybrid analysis. The 16S rRNA that surrounds 30S ribosomal protein S8 was identified by directed hydroxyl radical probing and then modeled into a 7.8 Å electron density map of the 70S ribosome. We also used directed hydroxyl radical probing of rRNA from the ribosome recycling factor (RRF) to determine its location and orientation in the ribosome.; The L-shaped structure of RRF bears a striking resemblance to tRNA and this structural similarity has suggested that the mode of action for RRF may be based on mimicry of tRNA. To determine whether or not RRF's interaction with the ribosome resembles that of tRNA, we probed rRNA in 70S ribosomes from Fe(II) tethered to ten different positions on the surface of RRF. Cleavage of 16S and 23S rRNA from the different probing positions constrained RRF to a well-defined location in the subunit interface cavity of the ribosome. Surprisingly, the orientation of RRF in the ribosome differed markedly from any of those previously observed for tRNA. Based on these results, we proposed a new mechanism for the function of RRF in ribosome recycling that is not based on straightforward mimicry of tRNA.