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  • 1. Bento-Abreu, Andre
    et al.
    Jager, Gunilla
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Swinnen, Bart
    Rué, Laura
    Hendrickx, Stijn
    Jones, Ashley
    Staats, Kim A.
    Taes, Ines
    Eykens, Caroline
    Nonneman, Annelies
    Nuyts, Rik
    Timmers, Mieke
    Silva, Lara
    Chariot, Alain
    Nguyen, Laurent
    Ravits, John
    Lemmens, Robin
    Cabooter, Deirdre
    Van Den Bosch, Ludo
    Van Damme, Philip
    Al-Chalabi, Ammar
    Bystrom, Anders
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Robberecht, Wim
    Elongator subunit 3 (ELP3) modifies ALS through tRNA modification.2018Ingår i: Human Molecular Genetics, ISSN 0964-6906, E-ISSN 1460-2083, Vol. 27, nr 7, s. 1276-1289Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Amyotrophic lateral sclerosis (ALS) is a fatal degenerative motor neuron disorder of which the progression is influenced by several disease-modifying factors. Here, we investigated ELP3, a subunit of the elongator complex that modifies tRNA wobble uridines, as one of such ALS disease modifiers. ELP3 attenuated the axonopathy of a mutant SOD1, as well as of a mutant C9orf72 ALS zebrafish model. Furthermore, the expression of ELP3 in the SOD1G93A mouse extended the survival and attenuated the denervation in this model. Depletion of ELP3 in vitro reduced the modified tRNA wobble uridine mcm5s2U and increased abundance of insoluble mutant SOD1, which was reverted by exogenous ELP3 expression. Interestingly, the expression of ELP3 in the motor cortex of ALS patients was reduced and correlated with mcm5s2U levels. Our results demonstrate that ELP3 is a modifier of ALS and suggest a link between tRNA modification and neurodegeneration.

  • 2. Chen, Peng
    et al.
    Jäger, Gunilla
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Zheng, Bo
    Transfer RNA modifications and genes for modifying enzymes in Arabidopsis thaliana2010Ingår i: BMC Plant Biology, ISSN 1471-2229, E-ISSN 1471-2229, Vol. 10, artikel-id 201Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Background: In all domains of life, transfer RNA (tRNA) molecules contain modified nucleosides. Modifications to tRNAs affect their coding capacity and influence codon-anticodon interactions. Nucleoside modification deficiencies have a diverse range of effects, from decreased virulence in bacteria, neural system disease in human, and gene expression and stress response changes in plants. The purpose of this study was to identify genes involved in tRNA modification in the model plant Arabidopsis thaliana, to understand the function of nucleoside modifications in plant growth and development. Results: In this study, we established a method for analyzing modified nucleosides in tRNAs from the model plant species, Arabidopsis thaliana and hybrid aspen (Populus tremula x tremuloides). 21 modified nucleosides in tRNAs were identified in both species. To identify the genes responsible for the plant tRNA modifications, we performed global analysis of the Arabidopsis genome for candidate genes. Based on the conserved domains of homologs in Sacccharomyces cerevisiae and Escherichia coli, more than 90 genes were predicted to encode tRNA modifying enzymes in the Arabidopsis genome. Transcript accumulation patterns for the genes in Arabidopsis and the phylogenetic distribution of the genes among different plant species were investigated. Transcripts for the majority of the Arabidopsis candidate genes were found to be most abundant in rosette leaves and shoot apices. Whereas most of the tRNA modifying gene families identified in the Arabidopsis genome was found to be present in other plant species, there was a big variation in the number of genes present for each family. Through a loss of function mutagenesis study, we identified five tRNA modification genes (AtTRM10, AtTRM11, AtTRM82, AtKTI12 and AtELP1) responsible for four specific modified nucleosides (m1G, m2G, m7G and ncm5U), respectively (two genes: AtKTI12 and AtELP1 identified for ncm5U modification). The AtTRM11 mutant exhibited an early-flowering phenotype, and the AtELP1 mutant had narrow leaves, reduced root growth, an aberrant silique shape and defects in the generation of secondary shoots. Conclusions: Using a reverse genetics approach, we successfully isolated and identified five tRNA modification genes in Arabidopsis thaliana. We conclude that the method established in this study will facilitate the identification of tRNA modification genes in a wide variety of plant species.

  • 3.
    Jäger, Gunilla
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Chen, Peng
    Björk, Glenn R.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Transfer RNA Bound to MnmH Protein Is Enriched with Geranylated tRNA - A Possible Intermediate in Its Selenation?2016Ingår i: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 11, nr 4, artikel-id e0153488Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The wobble nucleoside 5-methylaminomethyl-2-thio-uridine (mnm(5)s(2)U) is present in bacterial tRNAs specific for Lys and Glu and 5-carboxymethylaminomethyl-2-thio-uridine (cmnm(5)s(2)U) in tRNA specific for Gln. The sulfur of (c) mnm(5)s(2)U may be exchanged by selenium (Se)-a reaction catalyzed by the selenophosphate-dependent tRNA 2-selenouridine synthase encoded by the mnmH (ybbB, selU, sufY) gene. The MnmH protein has a rhodanese domain containing one catalytic Cys (C97) and a P-loop domain containing a Walker A motif, which is a potential nucleotide binding site. We have earlier isolated a mutant of Salmonella enterica, serovar Typhimurium with an alteration in the rhodanese domain of the MnmH protein (G67E) mediating the formation of modified nucleosides having a geranyl (ge)-group (C10H17-fragment) attached to the s(2) group of mnm(5)s(2)U and of cmnm(5)s(2)U in tRNA. To further characterize the structural requirements to increase the geranylation activity, we here report the analysis of 39 independently isolated mutants catalyzing the formation of mnm(5)ges(2)U. All these mutants have amino acid substitutions in the rhodanese domain demonstrating that this domain is pivotal to increase the geranylation activity. The wild type form of MnmH(+) also possesses geranyltransferase activity in vitro although only a small amount of the geranyl derivatives of (c) mnm(5)s(2)U is detected in vivo. The selenation activity in vivo has an absolute requirement for the catalytic Cys97 in the rhodanese domain whereas the geranylation activity does not. Clearly, MnmH has two distinct enzymatic activities for which the rhodanese domain is pivotal. An intact Walker motif in the P-loop domain is required for the geranylation activity implying that it is the binding site for geranylpyrophosphate (GePP), which is the donor molecule in vitro in the geranyltransfer reaction. Purified MnmH from wild type and from the MnmH(G67E) mutant have bound tRNA, which is enriched with geranylated tRNA. This in conjunction with earlier published data, suggests that this bound geranylated tRNA may be an intermediate in the selenation of the tRNA.

  • 4.
    Jäger, Gunilla
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Leipuviene, Ramune
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå centrum för molekylär patogenes (UCMP) (Teknisk-naturvetenskaplig fakultet).
    Pollard, Michael
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Qian, Qiang
    Björk, Glenn
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    The conserved Cys-X1-X2-Cys motif present in the TtcA protein is required for the thiolation of cytidine in position 32 of tRNA from Salmonella enterica serovar Typhimurium.2004Ingår i: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 186, nr 3, s. 750-757Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The modified nucleoside 2-thiocytidine (s(2)C) has so far been found in tRNA from organisms belonging to the phylogenetic domains Archaea and BACTERIA: In the bacteria Escherichia coli and Salmonella enterica serovar Typhimurium, s(2)C is present in position 32 of only four tRNA species-, and. An in-frame deletion of an S. enterica gene (designated ttcA, for "two-thio-cytidine") was constructed, and such a mutant has no detectable s(2)C in its tRNA. The TtcA protein family is characterized by the existence of both a PP-loop and a Cys-X(1)-X(2)-Cys motif in the central region of the protein but can be divided into two distinct groups based on the presence and location of additional Cys-X(1)-X(2)-Cys motifs in terminal regions of the sequence. Mutant analysis showed that both cysteines in this central conserved Cys-X(1)-X(2)-Cys motif are required for the formation of s(2)C. The DeltattcA1 mutant grows at the same rate as the congenic wild-type strain, and no growth disadvantage caused by the lack of s(2)C was observed in a mixed-population experiment. Lack of s(2)C32 did not reduce the selection rate at the ribosomal aminoacyl-tRNA site (A-site) for at any of its cognate CGN codons, whereas A-site selection at AGG by was dependent on the presence of s(2)C32. The presence of s(2)C32 in peptidyl- or in peptidyl- interfered with decoding in the A-site. The presence of s(2)C32 in decreased the rate of translation of the CGA codon but not that of the CGU codon.

  • 5.
    Jäger, Gunilla
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Nilsson, Kristina
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Björk, Glenn R.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    The Phenotype of Many Independently Isolated+1 Frameshift Suppressor Mutants Supports a Pivotal Role of the P-Site in Reading Frame Maintenance2013Ingår i: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, nr 4, s. e60246-Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The main features of translation are similar in all organisms on this planet and one important feature of it is the way the ribosome maintain the reading frame. We have earlier characterized several bacterial mutants defective in tRNA maturation and found that some of them correct a +1 frameshift mutation; i.e. such mutants possess an error in reading frame maintenance. Based on the analysis of the frameshifting phenotype of such mutants we proposed a pivotal role of the ribosomal grip of the peptidyl-tRNA to maintain the correct reading frame. To test the model in an unbiased way we first isolated many (467) independent mutants able to correct a +1 frameshift mutation and thereafter tested whether or not their frameshifting phenotypes were consistent with the model. These 467+1 frameshift suppressor mutants had alterations in 16 different loci of which 15 induced a defective tRNA by hypo- or hypermodifications or altering its primary sequence. All these alterations of tRNAs induce a frameshift error in the P-site to correct a +1 frameshift mutation consistent with the proposed model. Modifications next to and 39 of the anticodon (position 37), like 1-methylguanosine, are important for proper reading frame maintenance due to their interactions with components of the ribosomal P-site. Interestingly, two mutants had a defect in a locus (rpsI), which encodes ribosomal protein S9. The C-terminal of this protein contacts position 32-34 of the peptidyl-tRNA and is thus part of the P-site environment. The two rpsI mutants had a C-terminal truncated ribosomal protein S9 that destroys its interaction with the peptidyl-tRNA resulting in +1 shift in the reading frame. The isolation and characterization of the S9 mutants gave strong support of our model that the ribosomal grip of the peptidylt-RNA is pivotal for the reading frame maintenance.

  • 6. Kitamura, Aya
    et al.
    Nishimoto, Madoka
    Sengoku, Toru
    Shibata, Rie
    Jäger, Gunilla
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Björk, Glenn R.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Grosjean, Henri
    Yokoyama, Shigeyuki
    Bessho, Yoshitaka
    Characterization and Structure of the Aquifex aeolicus Protein DUF752 A BACTERIAL tRNA-METHYLTRANSFERASE (MnmC2) FUNCTIONING WITHOUT THE USUALLY FUSED OXIDASE DOMAIN (MnmC1)2012Ingår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 287, nr 52, s. 43950-43960Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Post-transcriptional modifications of the wobble uridine (U34) of tRNAs play a critical role in reading NNA/G codons belonging to split codon boxes. In a subset of Escherichia coli tRNA, this wobble uridine is modified to 5-methylaminomethyluridine (mnm5U34) through sequential enzymatic reactions. Uridine 34 is first converted to 5-carboxymethylaminomethyluridine (cmnm5U34) by the MnmE-Mnm Genzyme complex. The cmnm5U34 is further modified to mnm5U by the bifunctional MnmC protein. In the first reaction, the FAD-dependent oxidase domain (MnmC1) converts cmnm5U into 5-aminomethyluridine (nm5U34), and this reaction is immediately followed by the methylation of the free amino group into mnm5U34 by the S-adenosylmethionine-dependent domain (MnmC2). Aquifex aeolicus lacks a bifunctional MnmC protein fusion and instead encodes the Rossmann-fold protein DUF752, which is homologous to the methyltransferase MnmC2 domain of Escherichia coli MnmC (26% identity). Here, we determined the crystal structure of the A. aeolicus DUF752 protein at 2.5 Å resolution, which revealed that it catalyzes the S-adenosylmethionine-dependent methylation of nm5U in vitro, to form mnm5U34 in tRNA. We also showed that naturally occurring tRNA from A. aeolicus contains the 5-mnm group attached to the C5 atom of U34. Taken together, these results support the recent proposal of an alternative MnmC1-independent shortcut pathway for producing mnm5U34 in tRNAs.

  • 7. Koshla, Oksana
    et al.
    Yushchuk, Oleksandr
    Stash, Iryna
    Dacyuk, Yuriy
    Myronovskyi, Maksym
    Jäger, Gunilla
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Sussmuth, Roderich D.
    Luzhetskyy, Andriy
    Byström, Anders
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Kirsebom, Leif A.
    Ostash, Bohdan
    Gene miaA for post-transcriptional modification of tRNAXXA is important for morphological and metabolic differentiation in Streptomyces2019Ingår i: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 112, nr 1, s. 249-265Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Members of actinobacterial genus Streptomyces possess a sophisticated life cycle and are the deepest source of bioactive secondary metabolites. Although morphogenesis and secondary metabolism are subject to transcriptional co-regulation, streptomycetes employ an additional mechanism to initiate the aforementioned processes. This mechanism is based on delayed translation of rare leucyl codon UUA by the only cognate tRNA(UAA)(Leu) (encoded by bldA). The bldA-based genetic switch is an extensively documented example of translational regulation in Streptomyces. Yet, after five decades since the discovery of bldA, factors that shape its function and peculiar conditionality remained elusive. Here we address the hypothesis that post-transcriptional tRNA modifications play a role in tRNA-based mechanisms of translational control in Streptomyces. Particularly, we studied two Streptomyces albus J1074 genes, XNR_1074 (miaA) and XNR_1078 (miaB), encoding tRNA (adenosine(37)-N6)-dimethylallyltransferase and tRNA (N6-isopentenyl adenosine(37)-C2)-methylthiotransferase respectively. These enzymes produce, in a sequential manner, a hypermodified ms(2)i(6)A37 residue in most of the A36-A37-containing tRNAs. We show that miaB and especially miaA null mutant of S. albus possess altered morphogenesis and secondary metabolism. We provide genetic evidence that miaA deficiency impacts translational level of gene expression, most likely through impaired decoding of codons UXX and UUA in particular.

  • 8. Leitner, Johannes
    et al.
    Retzer, Katarzyna
    Malenica, Nenad
    Bartkeviciute, Rasa
    Lucyshyn, Doris
    Jäger, Gunilla
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Korbei, Barbara
    Byström, Anders
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Luschnig, Christian
    Meta-regulation of Arabidopsis Auxin Responses Depends on tRNA Maturation2015Ingår i: Cell reports, ISSN 2211-1247, E-ISSN 2211-1247, Vol. 11, nr 4, s. 516-526Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Polar transport of the phytohormone auxin throughout plants shapes morphogenesis and is subject to stringent and specific control. Here, we identify basic cellular activities connected to translational control of gene expression as sufficient to specify auxin-mediated development. Mutants in subunits of Arabidopsis Elongator, a protein complex modulating translational efficiency via maturation of tRNAs, exhibit defects in auxin-controlled developmental processes, associated with reduced abundance of PIN-formed (PIN) auxin transport proteins. Similar anomalies are observed upon interference with tRNA splicing by downregulation of RNA ligase (AtRNL), pointing to a general role of tRNA maturation in auxin signaling. Elongator Protein 6 (ELP6) and AtRNL expression patterns underline an involvement in adjusting PIN protein levels, whereas rescue of mutant defects by auxin indicates rate-limiting activities in auxin-controlled organogenesis. This emphasizes mechanisms in which auxin serves as a bottleneck for plant morphogenesis, translating common cellular activities into defined developmental readouts.

  • 9. Mehlgarten, Constance
    et al.
    Jablonowski, Daniel
    Wrackmeyer, Uta
    Tschitschmann, Susan
    Sondermann, David
    Jäger, Gunilla
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Gong, Zhizhong
    Byström, Anders S
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Schaffrath, Raffael
    Breunig, Karin D
    Elongator function in tRNA wobble uridine modification is conserved between yeast and plants2010Ingår i: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 76, nr 5, s. 1082-1094Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Based on studies in yeast and mammalian cells the Elongator complex has been implicated in functions as diverse as histone acetylation, polarized protein trafficking and tRNA modification. Here we show that Arabidopsis mutants lacking the Elongator subunit AtELP3/ELO3 have a defect in tRNA wobble uridine modification. Moreover, we demonstrate that yeast elp3 and elp1 mutants expressing the respective Arabidopsis Elongator homologues AtELP3/ELO3 and AtELP1/ELO2 assemble integer Elongator complexes indicating a high degree of structural conservation. Surprisingly, in vivo complementation studies based on Elongator-dependent tRNA nonsense suppression and zymocin tRNase toxin assays indicated that while AtELP1 rescued defects of a yeast elp1 mutant, the most conserved Elongator gene AtELP3, failed to complement an elp3 mutant. This lack of complementation is due to incompatibility with yeast ELP1 as coexpression of both plant genes in an elp1 elp3 yeast mutant restored Elongator’s tRNA modification function in vivo. Similarly, AtELP1, not ScELP1 also supported partial complementation by yeast-plant Elp3 hybrids suggesting that AtElp1 has less stringent sequence requirements for Elp3 than ScElp1. We conclude that yeast and plant Elongator share tRNA modification roles and propose that this function might be conserved in Elongator from all eukaryotic kingdoms of life.

  • 10.
    Nilsson, Kristina
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Jäger, Gunilla
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Björk, Glenn R.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    An unmodified wobble uridine in tRNAs specific for Glutamine, Lysine, and Glutamic acid from Salmonella enterica Serovar Typhimurium results in nonviability-Due to increased missense errors?2017Ingår i: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 12, nr 4, artikel-id e0175092Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    In the wobble position of tRNAs specific for Gln, Lys, and Glu a universally conserved 5-methylene- 2-thiouridine derivative (xm(5) s(2) U34, x denotes any of several chemical substituents and 34 denotes the wobble position) is present, which is 5-(carboxy) methylaminomethyl- 2-thiouridine ((c) mnm(5) s(2) U34) in Bacteria and 5-methylcarboxymethyl-2-thiouridine (mcm(5) s(2) U34) in Eukarya. Here we show that mutants of the bacterium Salmonella enterica Serovar Typhimurium LT2 lacking either the s(2) - or the (c) mnm(5) -group of (c) mnm(5) s(2) U34 grow poorly especially at low temperature and do not grow at all at 15 degrees C in both rich and glucose minimal media. A double mutant of S. enterica lacking both the s(2)- and the (c) mnm(5)-groups, and that thus has an unmodified uridine as wobble nucleoside, is nonviable at different temperatures. Overexpression of tRN(cmnm5s2UUG)(AGln) lacking either the s(2) - or the (c) mnm(5)-group and of tRNA(mnm5s2UUU)(Lys) lacking the s(2) -group exaggerated the reduced growth induced by the modification deficiency, whereas overexpression of tRNA(mnm5s2UUU)(Lys) lacking the mnm(5)-group did not. From these results we suggest that the primary function of cmnm(5) s(2) U34 in bacterial tRNA(cmnm5s2UUG)(Gln) and mnm(5) s(2) U34 in tRNA(Lys) (mnm5s2UUU) is to prevent missense errors, but the mnm(5) -group of tRNA(Lys) (mnm5s2UUU) does not. However, other translational errors causing the growth defect cannot be excluded. These results are in contrast to what is found in yeast, since overexpression of the corresponding hypomodified yeast tRNAs instead counteracts the modification deficient induced phenotypes. Accordingly, it was suggested that the primary function of mcm(5) s(2) U34 in these yeast tRNAs is to improve cognate codon reading rather than prevents missense errors. Thus, although the xm(5) s(2) U34 derivatives are universally conserved, their major functional impact on bacterial and eukaryotic tRNAs may be different.

  • 11.
    Urbonavicius, Jaunius
    et al.
    Umeå universitet, Teknisk-naturvetenskaplig fakultet, Molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Jäger, Gunilla
    Umeå universitet, Teknisk-naturvetenskaplig fakultet, Molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Björk, Glenn R
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Medicinska fakulteten).
    Amino acid residues of the Escherichia coli tRNA(m5U54)methyltransferase (TrmA) critical for stability, covalent binding of tRNA and enzymatic activity.2007Ingår i: Nucleic Acids Res, ISSN 1362-4962, Vol. 35, nr 10, s. 3297-305Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The Escherichia coli trmA gene encodes the tRNA(m5U54)methyltransferase, which catalyses the formation of m5U54 in tRNA. During the synthesis of m5U54, a covalent 62-kDa TrmA-tRNA intermediate is formed between the amino acid C324 of the enzyme and the 6-carbon of uracil. We have analysed the formation of this TrmA-tRNA intermediate and m5U54 in vivo, using mutants with altered TrmA. We show that the amino acids F188, Q190, G220, D299, R302, C324 and E358, conserved in the C-terminal catalytic domain of several RNA(m5U)methyltransferases of the COG2265 family, are important for the formation of the TrmA-tRNA intermediate and/or the enzymatic activity. These amino acids seem to have the same function as the ones present in the catalytic domain of RumA, whose structure is known, and which catalyses the formation of m5U in position 1939 of E. coli 23 S rRNA. We propose that the unusually high in vivo level of the TrmA-tRNA intermediate in wild-type cells may be due to a suboptimal cellular concentration of SAM, which is required to resolve this intermediate. Our results are consistent with the modular evolution of RNA(m5U)methyltransferases, in which the specificity of the enzymatic reaction is achieved by combining the conserved catalytic domain with different RNA-binding domains.

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