An Atomic Level Description of the Specific Interactions between Nascent Peptide and Ribosome Exit Tunnel

The ribosome, through highly coordinated and concerted movement, is responsible for the synthesis of all protein found within a cell.  In addition, it is so highly conserved that is can serve as a molecular time capsule, varying subtlety within the tree of life.  While new technology has led to an explosion of knowledge concerning the components of the ribosome, many questions remain.  One of these questions involves the nature of the exit tunnel and its level of interaction with a growing peptide before breeching the extraribosomal interface.  Through the use of peptolides (ketolide-peptide hybrids described previously), we report a level of interaction between varied peptide sequences and key residues within the exit tunnel.  This is being accomplished by cell-free translation inhibition assays followed by footprinting experiments of the ribosomal RNA (rRNA) through dimethyl sulfate (DMS) and 1-cyclohexyl-(2-morpholinoethyl)carbodiimide metho-p-toluene sulfonate (CMCT) modification in the presence of the peptolides.  Translation inhibition experiments, which showed that antibiotic activity of the peptolides remained intact (Table 1), have been finalized.  With the knowledge that the peptolides retain functionality, a series of primers and conditions have been optimized for footprinting experiments. Optimization was done for dideoxy termination (Sanger) sequencing, reverse transcription, as well as gel electrophoresis conditions (1).  The first interaction to target through footprinting was A2058 (E. coli numbering).  This key residue has been shown through numerous crystallographic models to serve as the binding site for macrolides and ketolides (2-6).  As shown in Figure 1, A2058 is strongly protected from modification by DMS by our peptolides.  Preliminary results are suggesting strong levels of interactions at multiple residues on the rRNA that can be seen in Figures 2 and 3.  Experimentation is ongoing to collect data for DMS and CMCT footprinting on all four primers, while looking for additional regions to explore by yet to be determined primers.



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