Molecular characterization of ribosomal E-site functions

The ribosome is a ribonucleoprotein complex consisting of ribosomal proteins and ribosomal RNAs. The primary function of the ribosome is to covalently link amino acids via peptide bonds into polypeptides. The substrates for this reaction (tRNA molecules) travel through three ribosomal binding sites A, P and E during protein synthesis. The function of the ribosomal E site is still poorly understood. It has been shown that two 2`-OH groups in the acceptor stem of tRNA at position 76 and 71 are crucial for the translocation from the P- into the E-site of the ribosome. Substitution of the 2`-OH at position 71 with 2`-F also showed clear translocation defects. Since 2`-fluorine can act as a H-bond acceptor it can be concluded that the 2`- OH at this residue functions as a H-bond donor. Equipped with the recent high resolution 70S crystal structures, potential interaction partners of these two crucial tRNA 2`-OH groups with 23S rRNA can now be experimentally characterized. Aim of this project is to use the already established gapped-cp-reconstitution system of 50S subunits to identify the critical interaction partners of these pivotal tRNA groups in the 23S rRNA on the molecular level. Modifications of 23S RNA residues will be introduced at promising residues into the backbone as well as into the nucleobases. Ideally we will be able to find the H-bond acceptor of the 2`-OH group of tRNA nucleotide 71 in the 23S rRNA and can then potentially reverse the pattern, by placing the H-donor into 23S RNA and the acceptor into position 71 of the tRNA. This would functionally establish the importance of such an interaction for tRNA movement from P- to E-site during translocation. To address these questions experimentally it is important to establish a reliable tRNA translocation assay employing ribosomes carrying gapped-cp-reconstituted 50S subunits. Initial experiments in the Polacek lab showed that toe- printing does work with ribosomes carrying in vitro reconstituted particles harboring full-length 23S rRNA transcripts. The next step on this line would be to optimize toe-printing conditions in such a way, that they would also work with 50S particles assembled from circularly permuted 23S rRNA. Alternatively, or in addition, we will try to set up an in vitro translocation assay employing fluorescently labeled mRNAs in analogy to published methods which would allow us to follow the translocation kinetics of chemically modified 50S more precisely compared to the toe-printing approach. Ultimately, in vitro translation reactions will be employed using gapped- cp-reconstituted 50S which can elucidate the functional significance of specific 23S rRNA functional groups in the E-site for protein synthesis.