(A) Specificity of the anti-GidA antibody. Equal amounts of total extracts of strains IC4639, IC5550 and IC5550 harbouring plasmid pIC1154 (where gidA is under control of P_tac promoter and LacI repressor) were subjected to western blot analysis using anti-GidA antibody. Strain IC5550/pIC1154 was grown in the absence of inducer IPTG, but there is escape of gidA expression. Molecular weight markers (on the left) are in kDa. (B) Subcellular localization of GidA. Western blot analysis with anti-GidA and anti-CyoA antibodies of cell fractions from strains IC4639 and IC5550 is shown. Equal amounts of bulk protein (50 μg) were loaded in each lane. T, total cell lysate; S, soluble fraction; M: membrane fraction.

GidA and MnmE interact with each other. (A) Western blot of GST pull down analysis using anti-GidA antibody. GST-MnmE or GST alone were used as baits for pull down analysis. Input: total lysate of strain IC5332 that was added to the corresponding bait previously immobilized to glutathione-agarose. Sup: fraction not retained by the resin-bound bait protein. El: the bound material that is eluted from the resin with glutathione buffer. Equivalent amounts of each fraction were loaded. (B) The same as in (A), but the baits were GST-GidA or GST, the lysate was from strain IC5287, and the western blot was developed with anti-MnmE antibody. (C) Coimmunopurification of FLAG-GidA and MnmE. FLAG-GidA was purified from strain DH5α/pIC1180 with anti-FLAG agarose as described in Materials and Methods, and the resulting fractions were analyzed by western blot using the antibodies indicated in the right side of each panel. Anti-EF-Tu was used as a negative control. The arrowhead marks MnmE position. The upper band detected in the MnmE western blot corresponds to an unrelated, unknown protein that is recognized by the anti-MnmE antibody and serves as a negative control. CL: cleared lysate of DH5α/pIC1180 (supernatant after sonication). Ins: pellet of cleared lysate. FT: flow through after incubation with anti-FLAG agarose. R: resin after washes. El 1: first eluate with elution buffer (free FLAG peptide). El 2: second eluate with elution buffer. (D) In vitro pull down analysis of purified recombinant MnmE (rMnmE) and FLAG-GidA proteins. After incubating both proteins together, anti-FLAG resin was added and fractions collected as described in Material and Methods. As a negative control, MnmE was processed without adding FLAG-GidA. Sup: supernatant after pelleting FLAG-agarose. El: proteins eluted with free FLAG peptide. Equivalent amounts of samples were loaded in each well. Size of molecular weight markers (MWM) is indicated on the left in kDa.

Spectral analysis of purified FLAG-GidA and mutant derivatives, to detect the presence of bound FAD. The UV-visible spectrum of the wild type, G13A, and G15A FLAG-GidA proteins is shown in panel A, D and E, respectively. Free FAD was used to obtain a reference spectrum (panel B), and rMnmE as a negative control (panel C). Insets: HPLC analysis of the GidA-bound cofactor (A) and FAD (B), which had retention times of 16.19 and 16.05 min, respectively. FMN had a retention time of 18.61 min (data not shown).

Western blot analysis of total extracts from strains MG1655 (wild-type) and IC5241 (gidA::Tn10) transformed with pJF119 or its derivatives pIC1154, pIC1177, pIC1178 and pIC1179 (carrying the wild-type, G13A, G15A and G13A/G15A gidA alleles, respectively), using anti-GidA antibody. The strains were grown during 2.5 h in LBT at 37°C with or without 1 mM IPTG, before processed. 50 μg of total proteins were loaded in each well. Size of the molecular weight marker (on the left) is in kDa.

Modification pathway of U34. Only steps relevant to this work are shown as understood. The first stage in the modification of U34 at position 5 is controlled by MnmE and GidA, but it is unclear how many steps precede the formation of cmnm⁵U. The cmnm⁵ group is then rearranged in two steps catalyzed by MnmC to synthesize the mnm⁵ side chain in some tRNAs. U34 may undergo thiolation at position 2 mediated by MnmA, IscS and other proteins (see text). It is worthy to mention that the mnmC gene is not evolutionarily conserved, which explains for the absence of mnm⁵s²U in yeast mitochondrial tRNAs.

Self-assembly of FLAG-GidA protein. (A) Purification of the recombinant protein FLAG-GidA. SDS–PAGE analysis of samples from different purification steps of the FLAG-GidA protein. The gel was stained with Coomassie blue. Lane 1, crude extract from uninduced DEV16/pIC1180; lane 2, crude extract from DEV16/pIC1180 induced with arabinose; lanes 3 and 4, cleared lysate (supernatant after sonication) and insoluble material (pellet after sonication) of the induced strain, respectively; lane 5, first eluate with elution buffer (containing free FLAG peptide); lane 6, second eluate with elution buffer. (B) Gel filtration analysis of purified FLAG-GidA. A total of 100 μg of FLAG-GidA were applied on a Superdex HR 200 column as described in Materials and Methods and 0.5 ml fractions collected. Markers indicate the positions of the standards: ferritin (440 kDa), β-amylase (224 kDa), aldolase (158 kDa), albumin (67 kDa). Inset: SDS–PAGE analysis of fractions indicated with asterisks in the chromatogram and designated as a, b, and c. (C) In vitro cross-linking of FLAG-GidA with DTSP. Protein samples, untreated or cross-linked with DTSP, were analysed by non-reducing SDS–PAGE. Lanes 4 and 5 show the effect of 2-ME on the reactions. Size of mass markers, run in lane 1, is indicated on the left in kDa.

GidA and MnmE form an α2β2 heterotetrameric complex. (A) Estimation of the molecular mass of the peak fractions in preparations of rMnmE, FLAG-GidA and a FLAG-GidA/rMnmE mix by gel-filtration chromatography. The major peaks from each preparation eluted at 149, 168 and 195 kDa, respectively. Markers indicate the positions of the standards: ferritin (440 kDa), β-amylase (224 kDa), aldolase (158 kDa), albumin (67 kDa). Elution fractions a to g from chromatography of the FLAG-GidA/MnmE mix were pooled for further analysis. (B) SDS–PAGE of elution fractions a to g from the FLAG-GidA/rMnmE mix chromatography showed in (A). Fractions (250 μl) were precipitated with trichloroacetic acid before loading. The gel was stained with Coomassie blue. Molecular masses are indicated on the right in kDa. (C) Native PAGE analysis of rMnmE, FLAG-GidA and a mix of FLAG-GidA/rMnmE. Lane 1: Molecular mass markers were β-amylase and albumin (monomer and dimer). Lane 2: 10 μg of rMnmE. Lane 3: 10 μg of FLAG-GidA. Lanes 4–6: 15, 30, and 40 μg, respectively, of total protein from a FLAG-GidA/rMnmE mix prepared as indicated in Materials and Methods. Positions of mass markers and their size in kilodaltons are indicated on the left. Positions of rMnmE, FLAG-GidA and FLAG-GidA•rMnmE specific complexes are indicated on the right, together with the apparent molecular mass of the complexes. (D) Native PAGE of fractions coming from gel-filtration of a FLAG-GidA/rMnmE mix. Lane 1: molecular mass markers were as (C), Lanes 2 and 3: elution fractions b and c, respectively, from an experiment like that shown in (A). Size of the mass markers and complexes are indicated on the right and left, respectively. Note correspondence between complexes detected here and those of (C).

HPLC chromatograms of tRNA hydrolysates. Strains used were: MG1655 (wild-type), IC5241 (gidA::Tn10), and IC5348 (mnmE::kan), in (A), and IC5241 transformed with plasmids pIC1154, pIC1177, pIC1178 and pIC1179 (carrying the wild-type, G13A, G15A and G13A/G15A gidA allele, respectively), in (B). Strains in (B) were grown with (+, right side) or without (−, left side) IPTG. The nucleosides were monitored at 314 nm to maximize the detection of thiolated nucleosides. mnm⁵s²U and s²U were identified by comparing UV spectra with published spectra (41). AU, absorbance units.

Models for the biosynthetic pathway leading to cmnm⁵U34, summarized from references 32 (A) and 9 (B).