Lysine 207 as the Site of Cross-linking between the 3′-End of Escherichia coli Initiator tRNA and Methionyl-tRNA Formyltransferase

Abstract

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 32 P-3-endlabeled E. coli initiator methionine tRNA with sodium metaperiodate and cross-linked it to MTF. The crosslinked 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 From assembly and packaging of RNA viruses (1) to mRNA localization during development (2), the specific recognition of RNAs by proteins plays an important role in many biological processes. Examples of these biological processes include RNA processing, RNA splicing, RNA transport, ribosome assembly, translation, and translational regulation (3). As molecules that interact with a variety of different proteins, tRNAs provide an excellent system for studying the molecular basis of specificity in recognition of RNAs by different proteins (4).We are studying the specific recognition of Escherichia coli initiator methionyl-tRNA (Met-tRNA), 1 during its formylation to formylmethionyl-tRNA by the enzyme methionyl-tRNA formyltransferase (MTF, EC 2.1.2.9; 10-formyltetrahydrofolate:L-methionyl-tRNA N-formyltransferase). Formylation of initiator Met-tRNA is important for initiation of protein synthesis in eubacteria and in eukaryotic organelles such as mitochondria and chloroplasts (5-9). In E. coli, formylation provides a positive determinant for allowing the initiation factor IF2 to select the initiator tRNA from other tRNAs (10, 11) and a second negative determinant for blocking the binding of elongation factor EF-Tu to the initiator tRNA (12-14). The formylation reaction is highly specific; the enzyme formylates the initiator Met-tRNA but not the elongator Met-tRNA or any other aminoacyl-tRNA (15). Previous studies have shown that most of the determinants on the initiator tRNA important for its formylation by MTF are clustered in the acceptor stem (16 -19) (Fig. 1). However, although the protein sequence of MTF is known (20), little is known about the amino acid residues in MTF that are important for this recognition and the molecular basis of the specificity in recognition.As a first step in identifying regions of MTF that come close to the acceptor stem of the tRNA, we have cross-linked periodate-oxidized tRNA to MTF and have analyzed the site(s) of cross-linking. We show that Lys 207 in the sequence KLSKE (207-211) of MTF is the site of cross-link to the 3Ј terminus of the tRNA.2 The cross-linking of periodate-oxidized E. coli tRNA fMet to MTF has been studied before by Blanquet and co-workers (21). However, the sites of cross-linking were not analyzed in the previous work. MATERIALS AND METHODSChemicals, Enzymes, and Radioisotopes-Sodium metaperiodate, folinic acid, carboxypeptidase Y (sequencing grade), and methionine were obtained from Sigma. Sod...

Mammalian Mitochondrial Methionyl-tRNA Transformylase from Bovine Liver

“…Secondary structure predictions using the ChouFasman or Robson methods (36, 37) have not able been to clearly identify a similar structure in MTF mt . The Lys at position 207 in the linker region between the two domains of E. coli MTF is thought to be involved in the interaction with the 3Ј-end of the tRNA (19). This residue (Lys-214) has been conserved in the spacer region between the domains of MTF mt (Fig.…”

“…3). The COOH-terminal domain of E. coli MTF is (19,35). The secondary structure of E. coli MTF is designated with red (␣-helix) and blue (␤-strand) lines above the sequence (35).…”

“…coli MTF is known to have some affinity for many different tRNAs that is mediated through an interaction between the 3Ј-end of the acceptor stem of the tRNA and regions surrounding Lys-207 in MTF (19) (Fig. 4).…”

“…There is no evidence that cytoplasmic tRNAs are imported into animal mitochondria (11). Thus, the single tRNA Met gene must, in some unknown manner, give rise to both an initiator tRNA (fMet-tRNA) and an elongator tRNA (Met-tRNA) (35,19).…”

“…Mitochondrial tRNA Met was purified by a solid-phase hybridization method using DNA probes complementary to the 30 bases at the 3Ј-end of the tRNA (16). E. coli tRNA fMet and tRNA mMet were purified as described (17,18 (19) and the Met-tRNAs were purified as described above.…”