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1.  The YqfN protein of Bacillus subtilis is the tRNA: m1A22 methyltransferase (TrmK) 
Nucleic Acids Research  2008;36(10):3252-3262.
N1-methylation of adenosine to m1A occurs in several different positions in tRNAs from various organisms. A methyl group at position N1 prevents Watson–Crick-type base pairing by adenosine and is therefore important for regulation of structure and stability of tRNA molecules. Thus far, only one family of genes encoding enzymes responsible for m1A methylation at position 58 has been identified, while other m1A methyltransferases (MTases) remain elusive. Here, we show that Bacillus subtilis open reading frame yqfN is necessary and sufficient for N1-adenosine methylation at position 22 of bacterial tRNA. Thus, we propose to rename YqfN as TrmK, according to the traditional nomenclature for bacterial tRNA MTases, or TrMet(m1A22) according to the nomenclature from the MODOMICS database of RNA modification enzymes. tRNAs purified from a ΔtrmK strain are a good substrate in vitro for the recombinant TrmK protein, which is sufficient for m1A methylation at position 22 as are tRNAs from Escherichia coli, which natively lacks m1A22. TrmK is conserved in Gram-positive bacteria and present in some Gram-negative bacteria, but its orthologs are apparently absent from archaea and eukaryota. Protein structure prediction indicates that the active site of TrmK does not resemble the active site of the m1A58 MTase TrmI, suggesting that these two enzymatic activities evolved independently.
PMCID: PMC2425500  PMID: 18420655
2.  Crystal structure of Bacillus subtilis TrmB, the tRNA (m7G46) methyltransferase 
Nucleic Acids Research  2006;34(6):1925-1934.
The structure of Bacillus subtilis TrmB (BsTrmB), the tRNA (m7G46) methyltransferase, was determined at a resolution of 2.1 Å. This is the first structure of a member of the TrmB family to be determined by X-ray crystallography. It reveals a unique variant of the Rossmann-fold methyltransferase (RFM) structure, with the N-terminal helix folded on the opposite site of the catalytic domain. The architecture of the active site and a computational docking model of BsTrmB in complex with the methyl group donor S-adenosyl-l-methionine and the tRNA substrate provide an explanation for results from mutagenesis studies of an orthologous enzyme from Escherichia coli (EcTrmB). However, unlike EcTrmB, BsTrmB is shown here to be dimeric both in the crystal and in solution. The dimer interface has a hydrophobic core and buries a potassium ion and five water molecules. The evolutionary analysis of the putative interface residues in the TrmB family suggests that homodimerization may be a specific feature of TrmBs from Bacilli, which may represent an early stage of evolution to an obligatory dimer.
PMCID: PMC1447647  PMID: 16600901
3.  Dihydropteridine Reductase as an Alternative to Dihydrofolate Reductase for Synthesis of Tetrahydrofolate in Thermus thermophilus 
Journal of Bacteriology  2004;186(2):351-355.
A strategy devised to isolate a gene coding for a dihydrofolate reductase from Thermus thermophilus DNA delivered only clones harboring instead a gene (the T. thermophilus dehydrogenase [DHTt] gene) coding for a dihydropteridine reductase which displays considerable dihydrofolate reductase activity (about 20% of the activity detected with 6,7-dimethyl-7,8-dihydropterine in the quinonoid form as a substrate). DHTt appears to account for the synthesis of tetrahydrofolate in this bacterium, since a classical dihydrofolate reductase gene could not be found in the recently determined genome nucleotide sequence (A. Henne, personal communication). The derived amino acid sequence displays most of the highly conserved cofactor and active-site residues present in enzymes of the short-chain dehydrogenase/reductase family. The enzyme has no pteridine-independent oxidoreductase activity, in contrast to Escherichia coli dihydropteridine reductase, and thus appears more similar to mammalian dihydropteridine reductases, which do not contain a flavin prosthetic group. We suggest that bifunctional dihydropteridine reductases may be responsible for the synthesis of tetrahydrofolate in other bacteria, as well as archaea, that have been reported to lack a classical dihydrofolate reductase but for which possible substitutes have not yet been identified.
PMCID: PMC305743  PMID: 14702303
4.  Genes of De Novo Pyrimidine Biosynthesis from the Hyperthermoacidophilic Crenarchaeote Sulfolobus acidocaldarius: Novel Organization in a Bipolar Operon 
Journal of Bacteriology  2002;184(16):4430-4441.
Sequencing a 8,519-bp segment of the Sulfolobus acidocaldarius genome revealed the existence of a tightly packed bipolar pyrimidine gene cluster encoding the enzymes of de novo UMP synthesis. The G+C content of 35.3% is comparable to that of the entire genome, but intergenic regions exhibit a considerably lower percentage of strong base pairs. Coding regions harbor the classical excess of purines on the coding strand, whereas intergenic regions do not show this bias. Reverse transcription-PCR and primer extension experiments demonstrated the existence of two polycistronic messengers, pyrEF-orf8 and pyrBI-orf1-pyrCD-orf2-orf3-orf4, initiated from a pair of divergent and partially overlapping promoters. The gene order and the grouping in two wings of a bipolar operon constitute a novel organization of pyr genes that also occurs in the recently determined genome sequences of Sulfolobus solfataricus P2 and Sulfolobus tokodaii strain 7; the configuration appears therefore characteristic of Sulfolobus. The quasi-leaderless pyrE and pyrB genes do not bear a Shine-Dalgarno sequence, whereas the initiation codon of promoter-distal genes is preceded at an appropriate distance by a sequence complementary to the 3′ end of 16S rRNA. The polycistronic nature of the pyr messengers and the existence of numerous overlaps between contiguous open reading frames suggests the existence of translational coupling. pyrB transcription was shown to be approximately twofold repressed in the presence of uracil. The mechanism underlying this modulation is as yet unknown, but it appears to be of a type different from the various attenuation-like mechanisms that regulate pyrB transcription in bacteria. In contrast, the pyrE-pyrB promoter/control region harbors direct repeats and imperfect palindromes reminiscent of target sites for the binding of a hypothetical regulatory protein(s).
PMCID: PMC135248  PMID: 12142413
5.  Purification and Characterization of Sa-Lrp, a DNA-Binding Protein from the Extreme Thermoacidophilic Archaeon Sulfolobus acidocaldarius Homologous to the Bacterial Global Transcriptional Regulator Lrp 
Journal of Bacteriology  2000;182(13):3661-3672.
Archaea, constituting the third primary domain of life, harbor a basal transcription apparatus of the eukaryotic type, whereas curiously, a large fraction of the potential transcription regulation factors appear to be of the bacterial type. To date, little information is available on these predicted regulators and on the intriguing interplay that necessarily has to occur with the transcription machinery. Here, we focus on Sa-lrp of the extremely thermoacidophilic crenarchaeote Sulfolobus acidocaldarius, encoding an archaeal homologue of the Escherichia coli leucine-responsive regulatory protein Lrp, a global transcriptional regulator and genome organizer. Sa-lrp was shown to produce a monocistronic mRNA that was more abundant in the stationary-growth phase and produced in smaller amounts in complex medium, this down regulation being leucine independent. We report on Sa-Lrp protein purification from S. acidocaldarius and from recombinant E. coli, both identified by N-terminal amino acid sequence determination. Recombinant Sa-Lrp was shown to be homotetrameric and to bind to its own control region; this binding proved to be leucine independent and was stimulated at high temperatures. Interference binding experiments suggested an important role for minor groove recognition in the Sa-Lrp–DNA complex formation, and mutant analysis indicated the importance for DNA binding of the potential helix-turn-helix motif present at the N terminus of Sa-Lrp. The DNA-binding capacity of purified Sa-Lrp was found to be more resistant to irreversible heat inactivation in the presence of l-leucine, suggesting a potential physiological role of the amino acid as a cofactor.
PMCID: PMC94536  PMID: 10850980

Results 1-5 (5)