Day 1 :
Dr. Akira Kaji received his PhD in Biological Sciences from Johns Hopkins University in 1958 and conducted postdoctoral research at the Rockefeller Institute, Vanderbilt School of Medicine, and the Oak Ridge National Laboratory. He obtained a faculty position at the University of Pennsylvania, which he still maintains. He has over 200 publications in reputed journals. He has two notable discoveries among his many contributions. First is the discovery of the binding of tRNA to the mRNA/ribosome complex, which contributed to Nirenberg’s breakthrough in the discovery of genetic code. Dr. Kaji also determined the fourth step of protein synthesis, ribosome recycling.
Ribosome recycling factor (RRF) is known to catalyze three separate reactions: release of tRNA, release of mRNA, and splitting of the ribosome. We used E. coli harboring temperature sensitive (ts) RRF and followed downstream reading of translationally coupled ORF. At the non-permissive temperature, ribosomes remained on the termination codon of the junction sequence (UAAUG) of coupled ORFs and translated downstream ORF. When upstream ORF was short, translation of the downstream reading was abolished, suggesting that the ribosomes released by RRF are moving toward the Shine-Dalgarno (SD) sequence of the upstream ORF. Thermal frame shift at the stop codon was also stopped by the upstream SD sequence. Our data suggest that the ribosome-bound mRNA may take a secondary structure around the junction sequence. For in vitro studies, we used mRNA that incorporated different radioactively labeled amino acids based on frameshift at the junction sequence of two translationally coupled ORFs. In the absence of RRF, the ribosome stayed on the mRNA and translated in frame with the termination codon UAA. In the presence of RRF, amino acid incorporation occurred in frame with the start codon AUG. This suggests that RRF releases the ribosome from UAA and the released ribosome binds to AUG and begins translation. With the use of tethered, unsplittable ribosomes (Ribo-T), we showed that complete ribosomal splitting is not required for the action of RRF in translational coupling. Therefore, the main role of RRF in translational coupling appears to be the release of ribosomes from mRNA.