Lysine 207 as the site of cross-linking between the 3'-end of Escherichia coli initiator tRNA and methionyl-tRNA formyltransferase.
The specific formylation of initiator methionyl-tRNA by methionyl-tRNA formyltransferase (MTF) is important for initiation of protein synthesis in Escherichia coli. In attempts to identify regions of MTF that come close to the 3'-end of the tRNA, we oxidized 32P-3'-end-labeled E. coli initiator methionine tRNA with sodium metaperiodate and cross-linked it to MTF. The cross-linked MTF was separated from uncross-linked MTF by DEAE-cellulose chromatography, and the tRNA in the cross-linked MTF was hydrolyzed with nuclease P1 and RNase T1, leaving behind an oxidized fragment of [32P]AMP attached to MTF. Trypsin digestion of the cross-linked MTF followed by high pressure liquid chromatography of the digest yielded two peaks of radioactive peptides, I* and II*. These peptides were characterized by N- and/or C-terminal sequencing and by matrix-assisted laser desorption ionization mass spectroscopy. Peptide I* contained amino acids Gln186-Lys210 with Lys207 as the site of the cross-link. Peptide II*, a partial digestion product, contained amino acids Gln186-Arg214 also with Lys207 as the site of the cross-link. The molecular masses of peptides I* and II* indicate that the final product of the cross-linking reaction between the periodate-oxidized AMP moiety of the tRNA and Lys207 is most likely a morpholino derivative rather than a reduced Schiff's base.
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Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
RajBhandary
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D000217:Acyltransferases / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D019877:Hydroxymethyl and Formyl Transferases; D008239:Lysine; D008969:Molecular Sequence Data; D012329:RNA, Bacterial / Q000378:metabolism*; D012358:RNA, Transfer, Met / Q000235:genetics / Q000378:metabolism*
D012329:RNA, Bacterial; D012358:RNA, Transfer, Met; D019877:Hydroxymethyl and Formyl Transferases; C021561:methionyl-tRNA formyltransferase; D000217:Acyltransferases; D008239:Lysine