【Structural and Functional Analysis of the Ribosomal Stalk Protein】
Translation is the process in which ribosomes link amino acids to synthesize proteins, based on the genetic information copied in mRNA. Translation is a complex reaction consisting of four stages: initiation, elongation, termination, and recycling, and proceeds by the interactions of the ribosome with individual translation factors that act in each stage. The ribosomal stalk protein is a long, flexible structure that exists in multiple copies on the large ribosomal subunit, and plays a crucial role in the interactions between the ribosome and various translation factors (Fig. 1). To date, we have found that the ribosomal stalk protein directly interacts with various GTPase and ATPase translators via its C-terminal region. We have also shown that the ribosome stalk protein is essential for recruiting translation factors to the ribosomal factor-binding center, and for the activities of individual translation factors on the ribosome.

However, the mechanisms by which the stalk protein interacts with various translation factors with different structures remain elusive. It is also unclear how the stalk protein recruits translation factors to the factor-binding center of the ribosome, and how it contributes to facilitating the actions of individual translation factors. To answer these open questions, we are performing X-ray crystallography and biochemical/molecular biological analyses (Fig. 2). We are conducting this project in collaboration with Dr. Toshio Uchiumi, Honorary Professor/Fellow of Niigata University (HP).

【Structural and Functional Analyses of Peptidyl-tRNA Hydrolase】
In translation, the peptide grows on the tRNA on the ribosome, and when the ribosome reaches a stop codon, the elongated peptide is released from the tRNA and translation normally terminates. However, due to amino acid starvation, mRNA decay, and other phenomena, the ribosome may stall and not reach the stop codon. In this case, stalled ribosomes release peptidyl-tRNAs, tRNAs with the elongating peptide remaining attached to them, as the premature products of protein synthesis. The accumulation of peptidyl-tRNAs is toxic because it depletes the pool of tRNAs available for translation, thereby halting protein synthesis. Peptidyl-tRNA hydrolase (Pth) is an enzyme that cleaves the ester bond between the peptide and the tRNA of the peptidyl-tRNA molecule, to recycle the tRNA for further rounds of protein synthesis (Fig. 3). Pth is present in all living organisms and is an essential protein in bacteria.

The amino acid and nucleotide sequences of peptidyl-tRNAs within cells vary, and Pth recognizes these diverse peptidyl-tRNA species as substrates. However, the mechanism for this sequence-independent recognition by Pth remains obscure. The catalytic process of the hydrolysis reaction is also unknown. Furthermore, it is unclear where Pth works in the cell. To answer these questions, we are using X-ray crystallography, biochemical/molecular biological analyses, and enzymatic analyses (Fig. 4). We are also performing molecular genetics research using yeast, in collaboration with Dr. Shuh-ichi Nishikawa of the Faculty of Science/Graduate School of Science and Technology, Niigata University (HP). Notably, Pth is in the spotlight as an attractive antibiotic target. The results of this project, therefore, will provide important data for the development research of new antibiotics.