Further insights into the tRNA modification process controlled by proteins MnmE and GidA of Escherichia coli

Abstract

In Escherichia coli, proteins GidA and MnmE are involved in the addition of the carboxymethylaminomethyl (cmnm) group onto uridine 34 (U34) of tRNAs decoding two-family box triplets. However, their precise role in the modification reaction remains undetermined. Here, we show that GidA is an FAD-binding protein and that mutagenesis of the N-terminal dinucleotide-binding motif of GidA, impairs capability of this protein to bind FAD and modify tRNA, resulting in defective cell growth. Thus, GidA may catalyse an FAD-dependent reaction that is required for production of cmnmU34. We also show that GidA and MnmE have identical cell location and that both proteins physically interact. Gel filtration and native PAGE experiments indicate that GidA, like MnmE, dimerizes and that GidA and MnmE directly assemble in an α2β2 heterotetrameric complex. Interestingly, high-performance liquid chromatography (HPLC) analysis shows that identical levels of the same undermodified form of U34 are present in tRNA hydrolysates from loss-of-function gidA and mnmE mutants. Moreover, these mutants exhibit similar phenotypic traits. Altogether, these results do not support previous proposals that activity of MnmE precedes that of GidA; rather, our data suggest that MnmE and GidA form a functional complex in which both proteins are interdependent.

YibK is the 2′-O-methyltransferase TrmL that modifies the wobble nucleotide in Escherichia coli tRNALeu isoacceptors

“…A slow-growth phenotype has previously been noted in E. coli mnmE and mnmG mutants that lack complete modification on the base of U34 in several tRNAs including tRNA Leu cmnm5UmAA (Yim et al 2006). Although it was feasible that lack of the ribose methylation at the same nucleotide might cause similar growth defects, no significant difference in the steady-state growth rate between the wild-type and the DyibK mutant was observed in rich medium at either 37°C (Table 4) or 42°C (17.1 6 0.3 and 18.0 6 0.7 min, respectively).…”

“…Björk (Landick et al 1990). P1 transduction (Miller 1990) was used to introduce the desired null allele into strain IC4639 (Yim et al 2006), a wild-type derivative from strain Dev16 (Elseviers et al 1984), IC5550, an mnmG::Tn10 derivative of IC4639 (Yim et al 2006), and BW25113. Correct insertion of mutations was checked by PCR using primers upstream-flanking the replaced gene and internal primers for the kan r gene (Datsenko and Wanner 2000) or mini Tn10 element.…”

Interactome analysis of longitudinal pharyngeal infection of cynomolgus macaques by group A Streptococcus

“…For example, the GAS thdF gene had an inverse correlation with PLG expression. Although little is known about the function of many of these GTPases, thdF catalyzes the modification of certain tRNA species and is required for proper base-pairing during codon-anticodon recognition (21,22). Inactivation of these genes induces frame-shifting and premature termination during translation (23), particularly affecting proteins with extensive regions of positive charge, such as DNA-binding transcription factors.…”

Bacillus subtilis exhibits MnmC-like tRNA modification activities

“…In particular, modified nucleosides of the anticodon loop transform the loop architecture and dynamics to meet the requirements that the ribosome places on all tRNAs [2,4,5]. In Escherichia coli , the MnmEG complex, formed by the dimeric proteins MnmE and MnmG [6], modifies the wobble uridine (U34) of tRNA Lys UUU , tRNA Glu UUC , tRNA Gln UUG , tRNA Leu UAA , tRNA Arg UCU , and tRNA Gly UCC [7,8]. MnmEG catalyzes the incorporation of either an aminomethyl (nm) or a carboxymethylaminomethyl (cmnm) group at position 5 of U34 using ammonium or glycine as substrate (Figure 1) [7].…”