title: C-terminal peptide appendix in a class I tRNA synthetase needed for acceptor-helix contacts and microhelix aminoacylation.
abstract: The 10 class I tRNA synthetases have an N-terminal nucleotide-binding fold which contains the catalytic center. Insertions into the nucleotide-binding fold provide contacts for acceptor-helix interactions, which stabilize the amino acid acceptor end of the tRNA substrate in the active site. A separate and largely nonconserved C-terminal domain provides contacts with distal parts of the tRNA, including the anticodon. For Escherichia coli methionyl tRNA synthetase, whose structure is known, the C-terminal domain is predominantly alpha-helical and forms a loop which interacts with the anticodon trinucleotide located about 76 A from the amino acid attachment site. Fused to the end of this helical domain is a peptide which curls back into the N-terminal nucleotide-binding fold and region of the active site. We show here that mutations in this peptide appendix disrupt aminoacylation and binding of a 7 base pair microhelix substrate based on the acceptor stem of tRNA(fMet), without affecting interactions with ATP or methionine or with the tRNA(fMet) anticodon. The impairment of acceptor-helix interactions by mutation of the C-terminal peptide can offset favorable anticodon interactions and severely reduce aminoacylation of tRNA(fMet). Thus, in addition to, or as an alternative to, acceptor-helix-binding insertions into the N-terminal nucleotide-binding fold, C-terminal peptide epitopes in some class I enzymes may provide a mechanism for facilitating RNA microhelix interactions with the catalytic site.
authors.affiliation: Department of Biology, Massachusetts Institute of Technology, Cambridge, 02139.
authors.forename: S
authors.initials: S
authors.lastname: Kim
authors.forename: J A
authors.initials: JA
authors.lastname: Landro
authors.forename: A J
authors.initials: AJ
authors.lastname: Gale
authors.forename: P
authors.initials: P
authors.lastname: Schimmel
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry; D001483:Base Sequence; D017931:DNA Primers / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology; D005816:Genetic Complementation Test; D006860:Hydrogen Bonding; D007700:Kinetics; D008718:Methionine-tRNA Ligase / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012358:RNA, Transfer, Met / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D046249:Transfer RNA Aminoacylation
chemical_list: D001426:Bacterial Proteins; D017931:DNA Primers; D012358:RNA, Transfer, Met; D008718:Methionine-tRNA Ligase