The hemK gene in Escherichia coli encodes the N(5)-glutamine methyltransferase that modifies peptide release factors

Abstract

Class 1 peptide release factors (RFs) in Escherichia coli are N(5)-methylated on the glutamine residue of the universally conserved GGQ motif. One other protein alone has been shown to contain N(5)-methylglutamine: E.coli ribosomal protein L3. We identify the L3 methyltransferase as YfcB and show that it methylates ribosomes from a yfcB strain in vitro, but not RF1 or RF2. HemK, a close orthologue of YfcB, is shown to methylate RF1 and RF2 in vitro. hemK is immediately downstream of and co-expressed with prfA. Its deletion in E.coli K12 leads to very poor growth on rich media and abolishes methylation of RF1. The activity of unmethylated RF2 from K12 strains is extremely low due to the cumulative effects of threonine at position 246, in place of alanine or serine present in all other bacterial RFs, and the lack of N(5)-methylation of Gln252. Fast-growing spontaneous revertants in hemK K12 strains contain the mutations Thr246Ala or Thr246Ser in RF2. HemK and YfcB are the first identified methyltransferases modifying glutamine, and are widely distributed in nature.

Fig. 1. Search for prmB in the vicinity of aroC. The pattern-matching application PATMAT (Wallace and Henikoff, 1992) was employed to locate coding sequences near aroC containing a potential glycine-rich AdoMet-binding motif 1 sequence. The pattern block used was derived from six protein MTases from E.coli. The highest score is associated with yfcB immediately upstream of aroC.

Fig. 2. Complementation of prmB cryosensitive growth. The prmB strain CL1447 (Colson et al., 1979) and its wild-type parental strain AB2557 were transformed with plasmids carrying a wild-type yfcB gene [pLV(yfcB⁺)], a mutant yfcB gene [pLV(yfcB^–)] or no insert (pLV1). The plate incubated at 25°C shows the cryosensitive growth of the prmB strain and complementation of the phenotype by plasmid pLV(yfcB⁺).

Fig. 3. Methylation of undermethylated ribosomal proteins by cell extracts enriched in YfcB. Ribosomes prepared from the prmB strain CL1447 (filled circles) or the wild-type parental strain AB2557 (open squares) were incubated with S-adenosyl-l-[methyl-³H]methionine and cell extract from cells overproducing YfcB. Control points (filled triangles) show the absence of methylation of undermethylated ribosomal proteins by a cell extract from the prmB strain CL1447. Each point corresponds to 12 pmol of ribosomes and 3 µg of cell extract protein (YfcB enriched) or 10 µg of cell extract (strain CL1447).

Fig. 4. Insertion of the His₆ tag and truncation of hemK by recombination with linear DNA. Redαβγ-stimulated recombination was used to insert a His₆ tag-coding sequence and tet^R-resistant cassette at the end of the prfA gene with or without truncation of hemK. The positions on the chromosome of the homologous terminal sequences allowing recombination are shown by boxes 1–3. PCR oligonucleotides used to make the inserts added the homologous sequences 1 and 3 so as to truncate hemK, or 1 and 2 so as to allow insertion without truncation and allow hemK expression by translation coupling to tetA.

Fig. 5. Methylation of RF1 and RF2 by HemK. Non-methylated RFs prepared by expression from overproducing vectors were incubated with S-adenosyl-l-[methyl-³H]methionine and cell extract from cells overproducing HemK: RF1 (filled circles), RF2Ala(His)₆ (open circles), RF2AlaGGE(His)₆ variant (open boxes). A control is shown with RF2Ala(His)₆ and a cell extract prepared from the ΔhemK strain SC8 (filled triangles). Each point corresponds to 25 pmol of added RF and 0.5 µg of cell extract protein (HemK enriched) or 20 µg of cell extract (strain SC8).

Fig. 6. Growth inhibition due to undermethylation of RF2(Thr246). (A) Wild-type K12 strain Xac was transformed by plasmid pLM2(RF2T246H6) overexpressing His-tagged RF2Thr246 and co-transformed with plasmid pW(hem11) expressing hemK or control plasmid pWSK129 without insert, showing that growth inhibition due to RF2Thr246 overproduction is suppressed by hemK expression. IPTG (1 mM) was present to induce RF2 and HemK expression. Control constructions shown are the double transformant with pW(hem11) and pLV1 parent plasmid without insert, and the double transformant with both parent plasmids without inserts, pWSK129 and pLV1. Transformants were streaked on LB-agar, 1 mM IPTG and incubated at 25°C. (B) HemK-truncated mutant strain SC5 was transformed with plasmid pLM2(RF2T246H6) expressing His-tagged RF2Thr246, plasmid pLM1(RF2A246H6) expressing His-tagged RF2Ala246 (lower two streaks) or control plasmid pLV1 without insert. Transformants were streaked on LB-Amp-Tet and incubated at 37°C.

Fig. 7. Amino acid sequence motifs in putative proteins of the HemK family and HemK-rel-arch families (InterPro signatures IPR004556/7). A search for conserved motifs using MEME (Bailey and Elkan, 1994) showed three clearly defined subfamilies. The motifs found are numbered in the order of identification by MEME. (A) The proteins characteristic of the subfamilies are shown in Table II. (B) The localization of each of the motifs 1–15 identified by MEME is shown superimposed on a typical member of each subfamily. The regions of the sequences including the common motifs 2, 5, 12 and 1 were aligned using Clustal (Thompson et al., 1994) and manually adjusted to remove small gaps from the MEME motifs.