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 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 (Fig. 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 anticodon-containing domain was added later in evolution. Similarly, the catalytic domain of the synthetase is thought to be the historical enzyme, which contains the active site and which interacts with the minihelix domain of the tRNA. 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 aimed at reconstructing the assembly of the synthetase-tRNA complexes in evolution.

Our recent studies have expanded on these concepts to elucidate a role for the tRNA in fine-structure recognition 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.

Publications

Alexander, R., Nordin, B., Schimmel, P. Activation of microhelix charging by localized helix destabilization. Proc. Natl. Acad. Sci. U.S.A. 95:12214, 1998.

Beuning, P.J., Yang, F., Schimmel, P., Musier-Forsyth, K. Specific atomic groups and RNA helix geometry in acceptor stem recognition by a tRNA synthetase. Proc. Natl. Acad. Sci. U.S.A. 94:10150, 1997.

Chihade, J., Hayashibara, K., Shiba, K., Schimmel, P. Strong selective pressure to mark an RNA acceptor stem for alanine. Biochemistry 37:9193, 1998.

Frugier, M., Schimmel, P. Subtle atomic group recognition in the RNA minor groove. Proc. Natl. Acad. Sci. U.S.A. 94:11291, 1997.

Glasfeld, E., Schimmel, P. Zinc-dependent tRNA binding by a peptide element within a tRNA synthetase. Biochemistry 37:6739, 1997.

Hale, S.P., Auld, D.S., Schmidt, E., Schimmel, P. Discrete nucleotides in tRNA for editing and aminoacylation. Science 276:1250, 1997.

Hale, S.P., Schimmel, P. DNA aptamer targets translational editing motif in a tRNA synthetase. Tetrahedron 53:11985, 1997.

Henderson, B.S., Beuning, P.J., Shi, J.-P., Bald, R., Fürste, J.P., Erdmann, V.A., Musier-Forsyth, K., Schimmel, P. Subtle functional interactions in the RNA minor groove at a nonessential base pair. J. Am. Chem. Soc. 120:9110, 1998.

Henderson, B.S., Schimmel, P. RNA-RNA interactions between oligonucleotide substrates for aminoacylation. Bioorg. Med. Chem. 5:1071, 1997.

Musier-Forsyth, K., Schimmel, P. Atomic determinants for aminoacylation of RNA minihelices and relationship to genetic code. Accounts Chem. Res., in press.

Nair, S., Ribas de Pouplana, L., Houman, F., Avruch, A., Shen, X., Schimmel, P. Species-specific tRNA recognition in relation to tRNA synthetase contact residues. J. Mol. Biol. 269:1, 1997.

Nureki, O., Vassylyev, D.G., Tateno, M., Shimada, A., Nakama, T., Fukai, S., Konno, M., Hendrickson, T.L., Schimmel, P., Yokoyama, S. Enzyme structure with two catalytic sites for double-sieve selection of substrate. Science 280:578, 1998.

Ribas de Pouplana, L., Beuchter, D., Sardesai, N.Y., Schimmel, P. Functional analysis of peptide motif for RNA microhelix binding suggests new family of RNA-binding domains. EMBO J. 17:5449, 1998.

Ribas de Pouplana, L., Schimmel, P. Reconstruction of quaternary structures of class II tRNA synthetases by rational mutagenesis of a conserved domain. Biochemistry 36:15041, 1997.

Ribas de Pouplana, L., Turner, R.J., Steer, B.A., Schimmel, P. Genetic code origins: tRNAs older than their synthetases? Proc. Natl. Acad. Sci. U.S.A. 95:11295, 1998.

Sardesai, N.Y., Schimmel, P. Noncovalent assembly of microhelix recognition by a class II tRNA synthetase. J. Am. Chem. Soc. 120:3269, 1998.

Schimmel, P., Alexander, R. All you need is RNA. Science 281:658, 1998.

Schimmel, P., Alexander, R. Diverse RNA substrates for aminoacylation: Clues to origins? Proc. Natl. Acad. Sci. U.S.A. 95:10351, 1998.

Schimmel, P., Söll, D. When protein engineering confronts the tRNA world. Proc. Natl. Acad. Sci. U.S.A. 94:10007, 1997.

Schimmel, P., Tao, J., Hill, J. Aminoacyl tRNA synthetases as targets for new anti-infectives. FASEB J., in press.

Sen, S., Zhou, H., Ripmaster, T., Hittleman, W.N., Schimmel, P., White, R.A. Expression of a gene encoding a tRNA synthetase-like protein is enhanced in tumorigenic human myeloid leukemia cells and is cell cycle stage- and differentiation-dependent. Proc. Natl. Acad. Sci. U.S.A. 94:6164, 1997.

Shiba, K., Hiromi, H., Schimmel, P. Maintaining genetic code through adaptations of tRNA synthetases to taxonomic domains. Trends Biochem. Sci. 22:453, 1997.

Shiba, K., Stello, T., Motegi, H., Noda, T., Musier-Forsyth, K., Schimmel, P. Human lysyl-tRNA synthetase accepts N73 variants and rescues E coli double-defective mutant. J. Biol. Chem. 272:22809, 1997.

Wakasugi, K., Quinn, C., Tao, N., Schimmel, P. Switching species-specific aminoacylation with a peptide transplant. EMBO J. 17:297, 1998.

Whelihan, E.F., Schimmel, P. Rescuing an essential protein-RNA complex with a nonessential appended domain. EMBO J. 16:2968, 1997.