New function of vitamin B12: cobamide-dependent reduction of epoxyqueuosine to queuosine in tRNAs of Escherichia coli and Salmonella typhimurium

Abstract

Queuosine (Q), 7-(((4,5-cis-dihydroxy-2-cyclopentene-1-yl)-amino)methyl)-7-deazaguanosine, and Q derivatives usually replace guanosine in the anticodon of tRNAsGUN of eubacteria and of cytoplasmic and mitochondrial tRNAs of lower and higher eucaryotes except yeasts. Q appears to be synthesized de novo exclusively in eubacteria, and the free-base queuine serves as a nutrient factor for eucaryotes. Recently, a Q derivative, oQ, containing a 2,3-epoxy-4,5-dihydroxycyclopentane ring, has been identified in Escherichia coli tRNATYF. Here we show that oQ is formed when E. coli or SalmoneUa typhimurium is grown in glucose-salt medium. The formation of oQ was independent of molecular oxygen, and oQ-tRNAs were converted to Q-tRNAs by adding cobalamin to the growth medium. Under strictly anaerobic conditions, considerable amounts of Q were present in E. coli and S. typhimurium tRNAs when the bacteria were grown in the presence of cobalt ions with glycerol as the carbon source and fumarate as the electron acceptor. Under these conditions, the biosynthesis of cobalamin was induced. The results suggest that oQ is derived from ribose and that oQ is finally reduced to Q by a cobamide-dependent enzyme.

The trmA promoter has regulatory features and sequence elements in common with the rRNA P1 promoter family of Escherichia coli

“…Gene expression of the BtuB polypeptide has been shown to be repressed when cells are grown in the presence of cobalamin (24). Interestingly, it has also been shown that vitamin B12 is involved in the modification of tRNA (14). We therefore mieasured the trmA gene activity in cells grown in different cobalamin concentrations as described by Kadner (24).…”

Structural analysis of the interaction of the tRNA modifying enzymes Tgt and QueA with a substrate tRNA

“…The purified tRNA transcript was analysed as described [6,10]. In short, the tRNA was hydrolysed with nuclease P1 (Boehringer Mannheim) and dephosphorylated with alkaline phosphatase from E. coli (Boehringer Mannheim).…”

Involvement of protein kinase C in the control of tRNA modification with queuine in HeLa cells

“…Although this modification at position 34 in the anticodon has been highly conserved in the course of evolution, only eubacteria can synthesize Q in tRNA de novo, involving unique S-adenosylmethionine and vitamin B 12 dependent reactions (4)(5)(6). Degradation of bacterial tRNAsQUN (Q-tRNAs) liberates the eukaryotic nutrition factor, queuine, the free base analogue of Q in tRNA (because queuine is present in eukaryotes as free base and as modified nucleoside in tRNA, we use the abbreviation 'q' or 'q-base' to denote the free base, and 'Q' for the modified nucleoside in tRNA).…”

“…The guanine acceptance is maximal when the tRNAs are completely Q-deficient (0% modification found in tRNA from HeLa cells grown in horse serum-supplemented medium), and is 0 when the tRNAs are completely converted to tRNAQUN (100% modification). (b) Cells were seeded as described under (a) and cultivated for 4 days with an exchange of the medium on the second day. Queuine alone (300 nM) or together with PDGF (final concentration 40 ng/ml; Biomol, Germany) and/or EGF (30 nM; Biomol, Germany) or with 12-O-tetradecanoylphorbol-13-acetate (TPA; 15 FM; Sigma, Germany) was added on day 4 without exchange of the medium and tRNA was isolated 24 h later.…”