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    • Albert Einstein College of Medicine: Doctoral Dissertations
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    • Albert Einstein College of Medicine (AECOM)
    • Albert Einstein College of Medicine: Doctoral Dissertations
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    PEPTIDES AT THE TRANSFER-RNA BINDING SITE OF ESCHERICHIA COLI METHIONYL - TRANSFER-RNA SYNTHETASE (PROTEIN SYNTHESIS, NUCLEIC ACIDS INTERACTIONS, LABELING, AFFINITY)

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    Date
    1985
    Author
    VALENZUELA, DARIO
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    Abstract
    The primary sequences and X-ray structures of E. coli tRNA('fMet) and of a well defined 64 KD tryptic fragment of E. coli methionyl-tRNA synthetase (MetRS) have been published. In order to determine the relative orientation of each macromolecule within the protein nucleic acid complex, a chemically reactive tRNA('fMet) derivative capable of stoichiometric coupling to the tRNA binding site of MetRS has been prepared.;The affinity labeling derivative of E. coli tRNA('fMet) carries an average of one reactive side chain per molecule, distributed over four structural regions. Each side chain contains a disulfide bond reactive towards cysteine residues and an N-hydroxysuccinimide ester group capable of coupling to lysine amino groups in proteins. Reaction of the derivatized tRNA with E. coli MetRS leads to crosslinking only by reaction with lysine residues in the protein. Examination of the tRNA present in the crosslinked complex reveals that the enzyme is coupled to side chains attached to the 5' terminal nucleotide, the dihydrouridine loop, the anticodon and the CCA sequence.;The procedure for purification of the peptides crosslinked to the tRNA was as follows. After tryptic digestion of the crosslinked tRNA('fMet)-MetRS complex, the peptides covalently attached to the tRNA were separated from the bulk of the free peptides by ethanol precipitation. Separation of the tRNA fraction from most of the remaining free peptides was achieved with an ion-exchange chromatography step under denaturing conditions. The tRNA-bound peptides were released from the tRNA by cleavage of the disulfide bond of the crosslinker and separated by reverse-phase HPLC.;Subnanomolar amino acid analysis of the HPLC fractions indicated that four of them had amino acid compositions expected from lysine peptides found in the known primary sequence of MetRS. The identity of these peptides have been confirmed by N-terminal microsequencing.;The identified peptides are crosslinked through the lysine residues at positions 402, 439, 465, and 640 in the primary sequence of MetRS. Lysines 402, 439, and 465 are also found in the biologically active tryptic fragments of MetRS (MTF). In the published biglobular crystal structure of MTF, the crosslinked peptides are part of the C-terminal globule. It has been reported by others that lysine 402 is protected from acetylation when tRNA('fMet) or tRNA(,m)('Met) is non-covalently bound to MetRS. In addition, the sequences surrounding lysine 402 are partially homologous to sequences present in yeast MetRS.;The data obtained in this study will be used to assist in the construction of a molecular model of the tRNA('fMet)-MetRS complex.
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    https://yulib002.mc.yu.edu/login?url=http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqm&rft_dat=xri:pqdiss:8601811
    https://hdl.handle.net/20.500.12202/3083
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    • Albert Einstein College of Medicine: Doctoral Dissertations [1674]

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