Novel non-specific DNA adenine methyltransferases

The mom gene of bacteriophage Mu encodes an enzyme that converts adenine to N6-(1-acetamido)-adenine in the phage DNA and thereby protects the viral genome from cleavage by a wide variety of restriction endonucleases. Mu-like prophage sequences present in Haemophilus influenzae Rd (FluMu), Neisseria meningitidis type A strain Z2491 (Pnme1) and H. influenzae biotype aegyptius ATCC 11116 do not possess a Mom-encoding gene. Instead, at the position occupied by mom in Mu they carry an unrelated gene that encodes a protein with homology to DNA adenine N6-methyltransferases (hin1523, nma1821, hia5, respectively). Products of the hin1523, hia5 and nma1821 genes modify adenine residues to N6-methyladenine, both in vitro and in vivo. All of these enzymes catalyzed extensive DNA methylation; most notably the Hia5 protein caused the methylation of 61% of the adenines in λ DNA. Kinetic analysis of oligonucleotide methylation suggests that all adenine residues in DNA, with the possible exception of poly(A)-tracts, constitute substrates for the Hia5 and Hin1523 enzymes. Their potential ‘sequence specificity’ could be summarized as AB or BA (where B = C, G or T). Plasmid DNA isolated from Escherichia coli cells overexpressing these novel DNA methyltransferases was resistant to cleavage by many restriction enzymes sensitive to adenine methylation

There is no freedom without responsibility

Analizujemy system organizacji nauki i jego zmiany w Polsce w ostatnim ćwierćwieczu, w kontekście zmian równolegle zachodzących na świecie. W oparciu o nasze doświadczenia z pracy w Międzynarodowym Instytucie Biologii Molekularnej i Komórkowej w Warszawie, ale także dzięki dobrym wzorcom z innych instytucji w Polsce i za granicą, przedstawiamy nasze przemyślenia w odpowiedzi na pytanie, jak rozwijać naukę w Polsce. Uważamy, że aby usprawnić system organizacji nauki w naszym kraju i szybko osiągnąć pozytywny efekt, należy skorzystać ze sprawdzonych wzorów z innych krajów oraz pozytywnych przykładów własnych polskich sukcesów. Realizacja tych zadań wymaga odważnych decyzji politycznych, które zapewnią naukowcom niezbędną do efektywnej pracy wolność, ale także powiążą tę wolność z odpowiedzialnością. Dzięki ludziom, którzy się tego podejmą i odpowiedzialnie wykorzystają oferowane możliwości, Polska będzie miała szansę na szybki postęp cywilizacyjny. Inwestycje w naukę są kluczowe dla rozwoju każdego kraju.We analyze the system of organization of science and its change in Poland in the last quarter century, in the context of parallel changes in the world. Based on our experience at the International Institute of Molecular and Cell Biology in Warsaw, but also thanks to good models from other institutions in Poland and abroad, we present our thoughts on the question of how to develop science in Poland. We think that in order to improve the system of science organization in our country and quickly achieve a positive effect, we must use proven models from other countries and positive examples of our own Polish successes. The implementation of these tasks requires courageous political decisions that will provide the scientists with the freedom necessary to work effectively, but will also bind this freedom with responsibility. Thanks to people, who take up this responsibility and take full advantage of the opportunities offered, Poland will have a chance for rapid civilization progress. Investments in science are crucial to the development of each country

Bioinformatics-guided identification and experimental characterization of novel RNA methyltransferases

info:eu-repo/semantics/publishe

The open reading frame TTC1157 of Thermus thermophilus HB27 encodes the methyltransferase forming N²-methylguanosine at position 6 in tRNA.

N(2)-methylguanosine (m(2)G) is found at position 6 in the acceptor stem of Thermus thermophilus tRNA(Phe). In this article, we describe the cloning, expression, and characterization of the T. thermophilus HB27 methyltransferase (MTase) encoded by the TTC1157 open reading frame that catalyzes the formation of this modified nucleoside. S-adenosyl-L-methionine is used as donor of the methyl group. The enzyme behaves as a monomer in solution. It contains an N-terminal THUMP domain predicted to bind RNA and contains a C-terminal Rossmann-fold methyltransferase (RFM) domain predicted to be responsible for catalysis. We propose to rename the TTC1157 gene trmN and the corresponding protein TrmN, according to the bacterial nomenclature of tRNA methyltransferases. Inactivation of the trmN gene in the T. thermophilus HB27 chromosome led to a total absence of m(2)G in tRNA but did not affect cell growth or the formation of other modified nucleosides in tRNA(Phe). Archaeal homologs of TrmN were identified and characterized. These proteins catalyze the same reaction as TrmN from T. thermophilus. Individual THUMP and RFM domains of PF1002 from Pyrococcus furiosus were produced. These separate domains were inactive and did not bind tRNA, reinforcing the idea that the THUMP domain acts in concert with the catalytic domain to target a particular position of the tRNA molecule.Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe

Crystal structures of the tRNA:m2G6 methyltransferase Trm14/TrmN from two domains of life

info:eu-repo/semantics/nonPublishe

Structure and function of the m2G6 methyltransferase Trm14/TrmN from two domains of life

info:eu-repo/semantics/nonPublishe

N2-methylation of guanosine at position 10 in tRNA is catalyzed by a THUMP domain-containing, S-adenosylmethionine-dependent methyltransferase, conserved in Archaea and Eukaryota.

In sequenced genomes, genes belonging to the cluster of orthologous group COG1041 are exclusively, and almost ubiquitously, found in Eukaryota and Archaea but never in Bacteria. The corresponding gene products exhibit a characteristic Rossmann fold, S-adenosylmethionine-dependent methyltransferase domain in the C terminus and a predicted RNA-binding THUMP (thiouridine synthases, RNA methyltransferases, and pseudouridine synthases) domain in the N terminus. Recombinant PAB1283 protein from the archaeon Pyrococcus abyssi GE5, a member of COG1041, was purified and shown to behave as a monomeric 39-kDa entity. This protein (EC 2.1.1.32), now renamed (Pab)Trm-G10, which is extremely thermostable, forms a 1:1 complex with tRNA and catalyzes the adenosylmethionine-dependent methylation of the exocyclic amino group (N(2)) of guanosine located at position 10. Depending on the experimental conditions used, as well as the tRNA substrate tested, the enzymatic reaction leads to the formation of either N(2)-monomethyl (m(2)G) or N(2)-dimethylguanosine (m(2)(2)G). Interestingly, (Pab)Trm-G10 exhibits different domain organization and different catalytic site architecture from another, earlier characterized, tRNA-dimethyltransferase from Pyrococcus furiosus ((Pfu)Trm-G26, also known as (Pfu)Trm1, a member of COG1867) that catalyzes an identical two-step dimethylation of guanosine but at position 26 in tRNAs and is also conserved among all sequenced Eukaryota and Archaea. The co-occurrence of these two guanosine dimethyltransferases in both Archaea and Eukaryota but not in Bacteria is a hallmark of distinct tRNAs maturation strategies between these domains of life.info:eu-repo/semantics/publishe

Sequence-structure-function analysis of the bifunctional enzyme MnmC that catalyses the last two steps in the biosynthesis of hypermodified nucleoside mnm5s2U in tRNA.

MnmC catalyses the last two steps in the biosynthesis of 5-methylaminomethyl-2-thiouridine (mnm(5)s(2)U) in tRNA. Previously, we reported that this bifunctional enzyme is encoded by the yfcK open reading frame in the Escherichia coli K12 genome. However, the mechanism of its activity, in particular the potential structural and functional dependence of the domains responsible for catalyzing the two modification reactions, remains unknown. With the aid of the protein fold-recognition method, we constructed a structural model of MnmC in complex with the ligands and target nucleosides and studied the role of individual amino acids and entire domains by site-directed and deletion mutagenesis, respectively. We found out that the N-terminal domain contains residues responsible for binding of the S-adenosylmethionine cofactor and catalyzing the methylation of nm(5)s(2)U to form mnm(5)s(2)U, while the C-terminal domain contains residues responsible for binding of the FAD cofactor. Further, point mutants with compromised activity of either domain can complement each other to restore a fully functional enzyme. Thus, in the conserved fusion protein MnmC, the individual domains retain independence as enzymes. Interestingly, the N-terminal domain is capable of independent folding, while the isolated C-terminal domain is incapable of folding on its own, a situation similar to the one reported recently for the rRNA modification enzyme RsmC.Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jFLWINinfo:eu-repo/semantics/publishe

The yggH gene of Escherichia coli encodes a tRNA (m7G46) methyltransferase

We cloned, expressed, and purified the Escherichia coli YggH protein and show that it catalyzes the S-adenosyl-L-methionine-dependent formation of N(7)-methylguanosine at position 46 (m(7)G46) in tRNA. Additionally, we generated an E. coli strain with a disrupted yggH gene and show that the mutant strain lacks tRNA (m(7)G46) methyltransferase activity.Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe

Sequence-structure-function relationships of a tRNA (m7G46) methyltransferase studied by homology modeling and site-directed mutagenesis.

The Escherichia coli TrmB protein and its Saccharomyces cerevisiae ortholog Trm8p catalyze the S-adenosyl-L-methionine-dependent formation of 7-methylguanosine at position 46 (m7G46) in tRNA. To learn more about the sequence-structure-function relationships of these enzymes we carried out a thorough bioinformatics analysis of the tRNA:m7G methyltransferase (MTase) family to predict sequence regions and individual amino acid residues that may be important for the interactions between the MTase and the tRNA substrate, in particular the target guanosine 46. We used site-directed mutagenesis to construct a series of alanine substitutions and tested the activity of the mutants to elucidate the catalytic and tRNA-recognition mechanism of TrmB. The functional analysis of the mutants, together with the homology model of the TrmB structure and the results of the phylogenetic analysis, revealed the crucial residues for the formation of the substrate-binding site and the catalytic center in tRNA:m7G MTases.Comparative StudyJournal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jFLWINinfo:eu-repo/semantics/publishe