| Class I peptide release factors 1 and 2 (RF1 and RF2) are responsible for accurate termination of protein synthesis by high fidelity recognition of stop codons in the decoding center of the ribosome. Analyses of crystal structures have revealed specific residues at the mRNA -- release factor interface that appear crucial for decoding center binding (Laurberg, et al. 2008; Wiexlbaumer, et al. 2008; Korostelev, et al. 2008). Due to intricate associations of class I release factors to the A-site, an addition class II release factor, RF3, is required for their removal (Freistroffer, Pavlov, et al. 1997).;A recently developed fluorescence based method to monitor decoding center interactions (Hetrick, Lee and Joseph 2009) was utilized to investigate the importance of histidine 197 (H197) in RF1 binding. The change in fluorescence of a pyrene probe attached to mRNA accurately determined the presence of RF1 in the A-site. H197 was found to be crucial for both efficient binding to the mRNA and orientation of the RF for catalysis of peptide release. RF1 was also found to bind in a biphasic manner. An adapted version of the assay was used to study the functional requirements of RF3 mediated removal of RF1. This proved useful in detecting RF1 release from the decoding center with RF3 and GTP, but the protein seemed to lack GTPase activity over basal ribosomal levels. In addition, no release was observed with GDPNP which was contrary to previous studies (Zavialov, Buckingham and Ehrenberg 2001; Zavialov, Mora, et al. 2002). |
