Hideko Kaji

Ribosome Recycling Factor (RRF) is an essential factor present in all living organisms except for Archea. In eukaryotes, RRF is localized in organelles and is essential for maintenance of the organelles. We created temperature sensitive yeast with temperature sensitive RRF in the mitochondria (Teyssier et al 2003).

RRF is a protein whose structure is a near perfect mimic of the tRNA structure (Selmer et al 1999). They are almost super imposable. RRF consists of domain I, corresponding to the anticodon portion of tRNA, and domain II, corresponding to the amino acid accepter region of tRNA. This has been confirmed with the RRF of other organisms.

RRF binds to the A/P-site of the post termination complex (PTC). At this site, RRF most closely approaches helix 71 and helix 69 of 23S rRNA with its universally conserved residues R129 and R132 (mutations of which confer lethal phenotypes). The binding site of RRF clashes with the A- and P-sites but not with the P/E-site where deacylated tRNA is situated. These conclusions were supported by recent data based on co-crystallization of 50S subunits with domain I of RRF (Wilson et al 2005). EF-G binds to the ribosome at the factor-binding site. We have proposed that this bound EF-G "translocates" (moves) RRF on the ribosome to the T-site (translocated site). Since the two domains of RRF are very flexible, we propose that this intra-molecular movement is important for RRF activity (Selmer et al 1999), a view shared by others.

The interaction of RRF with EF-G has been reported to be species specific based on the genetic swapping by others but our data with ec- and tt- factors, the species specificity is not completely withheld (Raj et al 2005), suggesting that genetic swapping in this case may not be valid.

Kinetic studies of RRF-ribosome interaction performed (Seo et al 2004) showed that the mRNA-nucleotide sequence surrounding the termination codon influences the ribosome's response to RRF. For example, when the border of two ORFs is UAAUG, the ribosome at the termination codon remains on the mRNA even with RRF present. However, in this case RRF functions to place the ribosome correctly at the initiation codon (Inokuchi et al 2000).

We obtained the first direct evidence that RRF is involved in the dissociation of vacant 70S ribosomes or PTC into subunits (Hirokawa et al 2005). The 70S ribosome that has completed one round of translation must be dissociated into subunits to reinitiate protein synthesis (Guthrie and Nomura, 1968). However, the mechanism of this crucial step has remained unknown till this year. Subramanian suggested that IF3 dissociates 70S ribosomes into subunits (Subramanian et al 1970). In contrast, Kaempfer (1972) claimed that IF3 is an anti-association factor. Although IF1 in cooperation with IF3 was reported to dissociate 70S ribosomes into subunits, this was observed only with high concentrations of IF3 or low concentrations of Mg2+ ion (Subamanian et al 1970). In a buffer made to simulate the in vivo condition with the added polyamines and 6 mM Mg2+, incomplete dissociation was observed in the presence of IF1 and IF3 (Hirokawa et al 2005).

Thus we postulate that the chain of events during the disassembly of PTC by these three factors is as follows: 1) RRF(ribosome recycling factor) binds to the PTC at the A/P-site of the ribosome. 2) EF-G (elongation factor G) binds to the factor-binding site on the ribosome. 3) RRF is moved by EF-G to the T-site (translocated site) on the ribosome accompanied by the release of P/E-site-bound tRNA. 4) The T-site bound RRF undergoes a conformational change by the action of EF-G. 5) Transient dissociation of 70S ribosomes into subunits takes place accompanied by the release of RRF and mRNA. 6) IF3 (initiation factor 3) binds to the dissociated 30S subunit leading to the stable dissociation of subunits (subunit formation detectable by SDGC (sucrose density gradient centrifugation). 7) The IF3-30S complex is now ready to engage in the initiation process for the new round of translation. Step 3) is similar to the conventional translocation but steps 4) and 5) differ from the translocation in that they strictly require GTP.

Other aspect of our effort is to develop anti-HIV & HTLV agents which inhibit these viruses at multiple steps of the viral life cycle. We have found that a) tetra deoxy- and ribo-guanylic acid (G4) act as anti-HIV and certain palindrome attachments to G4 enhanced anti-HIV effects, b)lignan compounds acts as potent anti-HIV agent, c) halogenated Gomisin J derivatives act as nonnucleotide inhibitors of HIV RT, and d) d- but not l-peptides of N-terminal half of CXCR4 showed higher biological stability with significant inhibitory activity in the replication of CXCR4-dependent HIV-1 strains. Elucidation of the mechanisms involved in these findings are attempted in a hope to develop more effective anti-HIV agents.

We are not only interested in protein synthesis but also degradation of proteins as we originally discovered arginylation of proteins via arginyl tRNA by arginyl tRNA protein transferase in 1963. This enzyme is found to have a vital function in the embryogenesis through cardiovascular development.

We are currently working on eukaryotic RRF and advancement of this aspect will aid further understandings of the function of RRF in all living cells.

Inhibition of bacterial as well as fungal RRF will be the ideal sites for future new antibiotic production since structual studies accumulated on RRF so far will provide clear directions to proceed in this endeavor.