News and Publications

Decoding Genetic Information in Translation

The genetic code was established more than 2 billion years ago and became universally 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 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 eukaryotic 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 (Fig. 1). 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 active as substrates (Figs. 1 and 2). 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.

A scheme for the assembly of the synthetase-tRNA complex in evolution is given in Figure 3. 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.

Our recent studies expanded on these concepts to elucidate 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 accuracy of the genetic code.

The significance of the tRNA L-shaped fold in evolution was highlighted by our recent finding of a structure-specific tRNA-binding protein in the extreme thermophile Aquifex aeolicus. The role of a structure-specific tRNA-binding motif in the evolutionary development of the contemporary systems of tRNA recognition is of great interest.

Publications

Alexander, R.W., Schimmel, P. Evidence for breaking domain-domain functional communication in a synthetase-tRNA complex. Biochemistry 38:16359, 1999.

Chihade, J.W., Schimmel, P. Assembly of a catalytic unit for RNA microhelix aminoacylation using nonspecific RNA binding domains. Proc. Natl. Acad. Sci. U. S. A. 96:12316, 1999.

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

Morales, A.J., Swairjo, M., Schimmel, P. Structure-specific tRNA binding protein from the extreme thermophile Aquifex aeolicus. EMBO J. 18:3475, 1999.

Musier-Forsyth, K., Schimmel, P. Atomic determinants for aminoacylation of RNA minihelices and relationship to genetic code. Accounts Chem. Res. 32:368, 1999.

Nomanbhoy, T.K., Hendrickson, T.L., Schimmel, P. Transfer RNA-dependent translocation of misactivated amino acids to prevent errors in protein synthesis. Mol. Cell 4:519, 1999.

Nordin, B., Schimmel, P. RNA determinants for translational editing: Mischarging a minihelix substrate by a tRNA synthetase. J. Biol. Chem. 274:6835, 199 9.

Nureki, O., Vassylyev, D.G., Tateno, M., Shimada, A., Nakama, T., Fukai, S., Konno, M., Hendrickson, T.L., Schimmel, P., Yokoyama, S. Proofreading by isoleucyl-transfer RNA synthetase: Response. Science 283:459a, 1999.

Sardesai, N.Y., Green, R., Schimmel, P. Efficient 50S ribosome-catalyzed peptide bond synthesis with an aminoacyl minihelix. Biochemistry 38:12080, 1999.

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., in press.

Schimmel, P. Aminoacyl tRNA synthetases and decoding of genetic information. In: The Encyclopedia of Molecular Biology. Creighton, T.E. (Ed.). Wiley, New York, 1999, Vol. 1, p. 111.

Schimmel, P. Translational editing. In: The Encyclopedia of Molecular Biology. Creighton, T.E. (Ed.). Wiley, New York, 1999, Vol. 4, p. 2642.

Schimmel, P., Ribas de Pouplana, L.l. Genetic code origins: Experiments confirm phylogenetic predictions and may explain puzzle. Proc. Natl. Acad. Sci. U. S. A. 96:327, 1999.

Schimmel, P., Wang, C.-C. Getting tRNA synthetases into the nucleus. Trends Biochem. Sci. 24:127, 1999.

Steer, B., Schimmel, P. Domain-domain communications in a miniature archaebacterial tRNA synthetase. Proc. Natl. Acad. Sci. U. S. A. 96:13644, 1999.

Steer, B.A., Schimmel, P. Different adaptations of the same peptide motif for tRNA functional contacts by closely homologous tRNA synthetases. Biochemistry 38:4965, 1999.

Steer, B.A., Schimmel, P. Major anticodon-binding region missing from an archaebacterial tRNA synthetase. J. Biol. Chem. 274:35601, 1999.

Wakasugi, K., Schimmel, P. Two distinct cytokines released from a human tRNA synthetase. Science 284:147, 1999.

Wang, C.-C., Schimmel, P. Species barrier to RNA recognition overcome with nonspecific RNA binding domains. J. Biol. Chem. 274:16508, 1999.