{'fraction_non_alphanumeric': 0.017857142857142856, 'fraction_numerical': 0.0007878151260504202, 'mean_word_length': 5.163430420711974, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Protein-RNA domain-domain interactions in a tRNA sythetase system

The aminoacyl-tRNA synthetases are a family of proteins that provide the enzymatic basis for the genetic code by catalyzing the transfer of an amino acid to its cognate tRNA. The synthetases are divided into two classes on the basis of conserved sequence motifs that correspond to two specific active site architectures. The focus of this work is the class I methionyl-tRNA synthetase (MetRS). All tRNAs adopt a similar L-shaped tertiary structure. This structure has two domains. One consists of the acceptor stem and the TYC stem and loop and the other consists of the dihydrouridine stem and loop and the anticodon stem and loop. Aminoacyl tRNA synthetases in both classes contain non-conserved domains in addition to the conserved active site domains. These non-conserved domains may have been aquired through the course of evolution in order to increase specificity and efficiency of aminoacylation. In the case of MetRS, the non-conserved C-terminal domain is predominantly oc-helical and contains residues that interact directly with the anticodon of the initiator methionine tRNA (tRNAfMet). The interactions of critical portions of each of the two major domains of tRNAfM et with MetRS and with one of its domains were studied in this work. Sub-pieces of the protein and tRNA were constructed for this purpose. The anticodon binding domain of E. coli methionyl-tRNA synthetase was fused to maltose binding protein. This fusion protein and the released, isolated domain are stable and have native-like structural characteristics as shown by circular dichroism and thermal denaturation studies. The fusion protein and the isolated domain bind specifically to a small RNA hairpin oligonucleotide that recapitulates the anticodon stem-loop of tRNAfMet, as shown by the technique of affinity coelectrophoresis. The binding specificity and affinity of the fusion protein and the C-terminal domain duplicate that of the interaction between native methionyl tRNA synthetase and the anticodon stem-loop oligonucleotide. Thus, the anticodon binding domain is functionally independent of the class defining catalytic core and can be joined to another protein with little change in RNA binding characteristics. The binding of a mutant MetRS variant to the individual domains of tRNAfMet was studied with RNA hairpin substrates that recapitulate the acceptor stem (microhelix). The dissociation constants of MetRS for wild-type and mutant microhelixfMet, as well as a noncognate microhelix, were determined. The combination of kinetic data for the aminoacylation of these microhelices and binding affinities for these microhelices established that specificity determinants in the acceptor stem of tRNAfMet affect discrimination primarily during the transition state of catalysis. The structures of transfer RNAs appear to be strained when bound to synthetases. Through our analysis of microhelix binding and anticodon stem-loop binding, as well as the binding of full length tRNAfMet, we were able estimate the free energy of strain for the binding of tRNAfMet to MetRS. Acceptor helix interactions occur primarily with the N-terminal catalytic domain. However, substitution of Arg533 to Ala in the C-terminal domain affected acceptor stem binding with little effect on anticodon stemloop binding. This mutation is located in a C-terminal peptide appendix that extends from the C-terminal domain of MetRS to fold back near the active site in the N-terminal domain. These experiments provide a direct demonstration of how acceptor helix interactions provide a functional connection between the two domains of the enzyme. Thesis Supervisor: Professor Paul Schimmel Title: John D. and Catherine T. MacArthur Professor of Biochemistry and Biophysics