Acquisition of a Stable Mutation in Mety Allows Efficient Initiation from an Amber Codon in Escherichia Coli

Escherichia coli strains harbouring elongator tRNAs that insert amino acids in response to a termination codon during elongation have been generated for various applications. Additionally, it was shown that expression of an initiator tRNA containinlog a CUA anticodon from a multicopy plasmid in E. coli resulted in initiation from an amber codon. Even though the initiation-based system remedies toxicity-related drawbacks, its usefulness has remained limited for want of a strain with a chromosomally encoded initiator tRNA 'suppressor'. E. coli K strains possess four initiator tRNA genes: the metZ, metW and metV genes, located at a single locus, encode tRNA_1^f^M^e^t, and a distantly located metY gene encodes a variant, tRNA_2^f^M^e^t. In this study, a stable strain of E. coli K-12 that affords efficient initiation from an amber initiation codon was isolated. Genetic analysis revealed that the metY gene in this strain acquired mutations to encode tRNA_2^f^M^e^t with a CUA anticodon (a U35A36 mutation). The acquisition of the mutations depended n the presence of a plasmid-borne copy of the mutant metY and recA+ host background. The mutations were observed when the plasmid-borne gene encoded tRNA_2^f^M^e^t (U35A36) with additional changes in the acceptor stem (G72; G72G73) but not in the anticodon stem (U29C30A31/U35A36/ \psi 39G40A41). The usefulness of this strain, and a possible role for multiple tRNA_1^f^M^e^t genes in E. coli in safeguarding their intactness, are discussed

An unexpected absence of queuosine modification in the tRNAs of an Escherichia coli B strain

The post-transcriptional processing of tRNAs decorates them with a number of modified bases important for their biological functions. Queuosine, found in the tRNAs with GUN anticodons (Asp, Asn, His, Tyr), is an extensively modified base whose biosynthetic pathway is still unclear. In this study, it was observed that the $tRNA^{Tyr}$ from Escherichia coli B105 (a B strain) migrated faster than that from E. coli CA274 (a K-12 strain) on acid urea gels. The organization of $tRNA^{Tyr}$ genes in E. coli B105 was found to be typical of the B strains. Subsequent analysis of $tRNA^{Tyr}$ and $tRNA^{His}$ from several strains of E. coli on acid urea gels, and modified base analysis of tRNA preparations enriched for $tRNA^{Tyr}$, showed that E. coli B105 lacked queuosine in its tRNAs. However, the lack of queuosine in tRNAs was not a common feature of all E. coli B strains. The tgt and queA genes in B105 were shown to be functional by their ability to complement tgt and queA mutant strains. These observations suggested a block at the step of the biosynthesis of $preQ_1$(or $preQ_0$) in the B105 strain. Interestingly, a multicopy vector harbouring a functional tgt gene was toxic to E. coli B105 but not to CA274. Also, in mixed cultures, E. coli B105 was readily competed out by the CA274 strain. The importance of these observations and this novel strain (E. coli B105) in unravelling the mechanism of $preQ_1$ or $preQ_0$ biosynthesis is discussed

Crystal structure of Rv2118c: an AdoMet-dependent methyltransferase from Mycobacterium tuberculosis H37Rv

Rv2118c belongs to the class of conserved hypothetical proteins from Mycobacterium tuberculosis H37Rv. The crystal structure of Rv2118c in complex with S-adenosyl-Image -methionine (AdoMet) has been determined at 1.98 Å resolution. The crystallographic asymmetric unit consists of a monomer, but symmetry-related subunits interact extensively, leading to a tetrameric structure. The structure of the monomer can be divided functionally into two domains: the larger catalytic C-terminal domain that binds the cofactor AdoMet and is involved in the transfer of methyl group from AdoMet to the substrate and a smaller N-terminal domain. The structure of the catalytic domain is very similar to that of other AdoMet-dependent methyltransferases. The N-terminal domain is primarily a β-structure with a fold not found in other methyltransferases of known structure. Database searches reveal a conserved family of Rv2118c-like proteins from various organisms. Multiple sequence alignments show several regions of high sequence similarity (motifs) in this family of proteins. Structure analysis and homology to yeast Gcd14p suggest that Rv2118c could be an RNA methyltransferase, but further studies are required to establish its functional role conclusively