News and Publications

Decoding Genetic Information in Translation

The genetic code was established more than 2 billion years ago and became universally a part of all living organisms. The rules of the code, which relate specific nucleotide triplets to specific amino acids, are determined by aminoacylation reactions catalyzed by aminoacyl tRNA synthetases. In these reactions, an amino acid is associated with a specific nucleotide triplet of the genetic code by virtue of being linked to a specific tRNA that harbors the anticodon triplet cognate to the amino acid. Because of their central role in establishing the rules of the code, the tRNAs are thought to have arisen quite early, perhaps in the context of an RNA world. The synthetases may have been among the earliest proteins to appear, perhaps replacing ribozymes that catalyzed the aminoacylation of primordial tRNAs.

We wish to understand all aspects of these systems. Areas of research include detailed studies of molecular recognition of RNA, as represented by the synthetase-tRNA interactions and by peptide models of those interactions; editing and correction of potential errors in translation, as represented by the misacylation of tRNAs and the misactivation of amino acids; reconstruction of the assembly of tRNA through domain-domain interactions of RNA pieces; and dissection of catalytic and RNA-binding components of tRNA synthetases.

We are also interested in evolutionary relationships between synthetases that can reveal insight into the origins of the code and protein synthesis; involvement of eukaryote synthetases in higher order cellular functions, such as cell signaling pathways and the construction of macromolecular assemblies; and design and synthesis of systems of tRNA-like molecules that can be acylated and assemble complexes for peptide synthesis, as examples of the potential for early systems of protein synthesis that predated the ribosome.

The cloverleaf structure of tRNA is folded into 2 domains. One domain contains the anticodon with the template-reading head of the genetic code; the other, called the minihelix domain, contains the amino acid attachment site. The minihelix domain itself is a substrate for aminoacylation for at least 10 of the synthetases. Pieces even smaller than the minihelix are also active as substrates, as are special constructs known as pseudoknots (Fig. 1). Because these pieces lack the anticodon trinucleotides, the relationship between the sequences and structures of these active pieces (with as few as 4 bp) and the specific amino acid is distinct from the genetic code. The relationship between sequences and structures in tRNA acceptor stems and specific amino acids is referred to as an operational RNA code for amino acids. The operational RNA code is thought to have predated the genetic code.

Each domain of the synthetase interacts with a separate domain of the tRNA. The minihelix domain of the tRNA contains the amino acid attachment site and is thought to be the earliest version of a tRNA molecule. The domain containing the anticodon was added later in evolution. Similarly, the catalytic domain of the synthetase, which contains the active site and which interacts with the minihelix domain of the tRNA, is thought to be the historical enzyme. Later, a second domain of the synthetase was added; this domain interacts with the second domain of the tRNA. This model is useful for guiding experiments that aim at reconstructing the assembly of the synthetase-tRNA complexes in evolution.

Recently, we elucidated a role for the tRNA in recognition of the fine structure of amino acids. That is, the ability to distinguish between 2 closely similar amino acids is greatly enhanced by an effector function of the tRNA, which causes the rejection of amino acids that are not exactly matched with the synthetase and its cognate tRNA. This RNA-dependent fine-structure recognition may have developed first in an RNA world and later was incorporated into the synthetase system as a critical part of maintaining the genetic code.

Our recent investigations forged a link between protein synthesis and signal transduction pathways in human cells. Specifically, 2 distinct cytokines can be released from human tyrosyl-tRNA synthetase. The enzyme is secreted during apoptosis in cell culture (Fig. 2). After secretion, it can be split into 2 fragments by an extracellular protease such as leukocyte elastase. Each of the 2 fragments is a distinct cytokine (the unsplit, native enzyme has no cytokine activity). For example, the C-terminal fragment has potent leukocyte and monocyte chemotaxis activity and triggers production of tissue factor, myeloperoxidase, and tumor necrosis factor-α. The N-terminal fragment has the activities of IL-8. These results suggest applications to medicine in the areas of inflammation and cancer.

Publications

Chihade, J.W., Brown, J., Schimmel, P., Ribas de Pouplana, L. Origin of mitochondria in relation to evolutionary history of eukaryotic alanyl-tRNA synthetase. Proc. Natl. Acad. Sci. U. S. A. 97:12153, 2000.

Farrow, M.A., Nordin, B.E., Schimmel, P. Nucleotide determinants for tRNA-dependent amino acid discrimination by a class I tRNA synthetase. Biochemistry 38:16898, 1999.

Hendrickson, T.L., Nomanbhoy, T.K., Schimmel, P. Errors from selective disruption of the editing center in a tRNA synthetase. Biochemistry 39:8180, 2000.

Houman, F., Rho, S.B., Zhang, J., Shen. X., Wang, C.-C., Schimmel, P., Martinis, S.A. A prokaryote and human tRNA synthetase provide an essential RNA splicing function in yeast mitochondria. Proc. Natl. Acad. Sci. U. S. A. 97:13743, 2000.

Kelley, S.O., Steinberg, S.V., Schimmel, P. Functional defects of pathogenic human mitochondrial tRNAs related to structural fragility. Nat. Struct. Biol. 7:862, 2000.

Nomanbhoy, T.K., Schimmel, P. Misactivated amino acids translocate at similar rates across the surface of a tRNA synthetase. Proc. Natl. Acad. Sci. U. S. A. 97:5119, 2000.

Ribas de Pouplana, L., Schimmel, P. A view into the origin of life: Aminoacyl tRNA synthetases. Cell. Mol. Life Sci. 57:865, 2000.

Sardesai, N.Y., Stagg, S.M., VanLoock, M.S., Harvey, S.C., Schimmel, P. RNA scaffolds for minihelix-based aminoacyl transfer: Design of "transpeptizymes." J. Biomol. Stereodyn. II:29, 2000.

Schimmel, P., Kelley, S.O. Exiting an RNA world. Nat. Struct. Biol. 7:5, 2000.

Schimmel, P., Ribas de Pouplana, L. Footprints of aminoacyl tRNA synthetases are everywhere. Trends Biochem. Sci. 25:207, 2000.

Swairjo, M.A., Morales, A.J., Wang, C.-C., Ortiz, A.R., Schimmel, P. Crystal structure of Trbp111: A structure-specific tRNA binding protein. EMBO J. 19:6287, 2000.

Tao, J., Wendler, P., Connelly, G., Lim, A., Zhang, J., King, M., Li, T., Silverman, J.A., Schimmel, P., Tally, F.P. Drug target validation: Lethal infection blocked by inducible peptide. Proc. Natl. Acad. Sci. U. S. A. 97:783, 1999.

Wakasugi, K., Schimmel, P. Highly differentiated motifs responsible for two cytokine activities of a split human tRNA synthetase. J. Biol. Chem. 274:23155, 1999.

Wang, C.-C., Morales, A.J., Schimmel, P. Functional redundancy in the nonspecific RNA binding domain of a class I tRNA synthetase. J. Biol. Chem. 275:17180, 2000.