umu.sePublications
Change search
Refine search result
1 - 41 of 41
CiteExportLink to result list
Permanent link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Rows per page
  • 5
  • 10
  • 20
  • 50
  • 100
  • 250
Sort
  • Standard (Relevance)
  • Author A-Ö
  • Author Ö-A
  • Title A-Ö
  • Title Ö-A
  • Publication type A-Ö
  • Publication type Ö-A
  • Issued (Oldest first)
  • Issued (Newest first)
  • Created (Oldest first)
  • Created (Newest first)
  • Last updated (Oldest first)
  • Last updated (Newest first)
  • Disputation date (earliest first)
  • Disputation date (latest first)
  • Standard (Relevance)
  • Author A-Ö
  • Author Ö-A
  • Title A-Ö
  • Title Ö-A
  • Publication type A-Ö
  • Publication type Ö-A
  • Issued (Oldest first)
  • Issued (Newest first)
  • Created (Oldest first)
  • Created (Newest first)
  • Last updated (Oldest first)
  • Last updated (Newest first)
  • Disputation date (earliest first)
  • Disputation date (latest first)
Select
The maximal number of hits you can export is 250. When you want to export more records please use the Create feeds function.
  • 1. Atkins, John F
    et al.
    Björk, Glenn R
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    A gripping tale of ribosomal frameshifting: extragenic suppressors of frameshift mutations spotlight P-site realignment2009In: Microbiology and molecular biology reviews, ISSN 1092-2172, E-ISSN 1098-5557, Vol. 73, no 1, p. 178-210Article in journal (Refereed)
    Abstract [en]

    Mutants of translation components which compensate for both -1 and +1 frameshift mutations showed the first evidence for framing malleability. Those compensatory mutants isolated in bacteria and yeast with altered tRNA or protein factors are reviewed here and are considered to primarily cause altered P-site realignment and not altered translocation. Though the first sequenced tRNA mutant which suppressed a +1 frameshift mutation had an extra base in its anticodon loop and led to a textbook "yardstick" model in which the number of anticodon bases determines codon size, this model has long been discounted, although not by all. Accordingly, the reviewed data suggest that reading frame maintenance and translocation are two distinct features of the ribosome. None of the -1 tRNA suppressors have anticodon loops with fewer than the standard seven nucleotides. Many of the tRNA mutants potentially affect tRNA bending and/or stability and can be used for functional assays, and one has the conserved C74 of the 3' CCA substituted. The effect of tRNA modification deficiencies on framing has been particularly informative. The properties of some mutants suggest the use of alternative tRNA anticodon loop stack conformations by individual tRNAs in one translation cycle. The mutant proteins range from defective release factors with delayed decoding of A-site stop codons facilitating P-site frameshifting to altered EF-Tu/EF1alpha to mutant ribosomal large- and small-subunit proteins L9 and S9. Their study is revealing how mRNA slippage is restrained except where it is programmed to occur and be utilized.

  • 2.
    Björk, Glenn
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Transfer RNA modification2005In: EcoSal - Escherichia coli and Salmonella. Cellular and Molecular Biology, ASM Press, Washington DC , 2005Chapter in book (Refereed)
  • 3.
    Björk, Glenn
    et al.
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Huang, Bo
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Persson, Olof P
    Byström, Anders
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    A conserved modified wobble nucleoside (mcm5s2U) in lysyl-tRNA is required for viability in yeast.2007In: RNA, ISSN 1355-8382, Vol. 13, no 8, p. 1245-55Article in journal (Refereed)
    Abstract [en]

    Transfer RNAs specific for Gln, Lys, and Glu from all organisms (except Mycoplasma) and organelles have a 2-thiouridine derivative (xm(5)s(2)U) as wobble nucleoside. These tRNAs read the A- and G-ending codons in the split codon boxes His/Gln, Asn/Lys, and Asp/Glu. In eukaryotic cytoplasmic tRNAs the conserved constituent (xm(5)-) in position 5 of uridine is 5-methoxycarbonylmethyl (mcm(5)). A protein (Tuc1p) from yeast resembling the bacterial protein TtcA, which is required for the synthesis of 2-thiocytidine in position 32 of the tRNA, was shown instead to be required for the synthesis of 2-thiouridine in the wobble position (position 34). Apparently, an ancient member of the TtcA family has evolved to thiolate U34 in tRNAs of organisms from the domains Eukarya and Archaea. Deletion of the TUC1 gene together with a deletion of the ELP3 gene, which results in the lack of the mcm(5) side chain, removes all modifications from the wobble uridine derivatives of the cytoplasmic tRNAs specific for Gln, Lys, and Glu, and is lethal to the cell. Since excess of the unmodified form of these three tRNAs rescued the double mutant elp3 tuc1, the primary function of mcm(5)s(2)U34 seems to be to improve the efficiency to read the cognate codons rather than to prevent mis-sense errors. Surprisingly, overexpression of the mcm(5)s(2)U-lacking tRNA(Lys) alone was sufficient to restore viability of the double mutant.

  • 4.
    Björk, Glenn R.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Adventures with Frameshift Supressor tRNAs2011In: Lure of Bacterial Genetics: a Tribute to John Roth / [ed] Stanley Maloy, Kelly T. Hughes and Josep Casadesús, WASHINGTON: American society for microbiology, ASM Press , 2011, p. 131-140Chapter in book (Refereed)
  • 5.
    Björk, Glenn R
    et al.
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine).
    Jacobsson, Kerstin
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Nilsson, Kristina
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Johansson, Marcus J O
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Byström, Anders S
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Persson, Olof P
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    A primordial tRNA modification required for the evolution of life?2001In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 20, no 1-2, p. 231-239Article in journal (Refereed)
    Abstract [en]

    The evolution of reading frame maintenance must have been an early event, and presumably preceded the emergence of the three domains Archaea, Bacteria and Eukarya. Features evolved early in reading frame maintenance may still exist in present-day organisms. We show that one such feature may be the modified nucleoside 1-methylguanosine (m(1)G37), which prevents frameshifting and is present adjacent to and 3' of the anticodon (position 37) in the same subset of tRNAs from all organisms, including that with the smallest sequenced genome (Mycoplasma genitalium), and organelles. We have identified the genes encoding the enzyme tRNA(m(1)G37)methyltransferase from all three domains. We also show that they are orthologues, and suggest that they originated from a primordial gene. Lack of m(1)G37 severely impairs the growth of a bacterium and a eukaryote to a similar degree. Yeast tRNA(m(1)G37)methyltransferase also synthesizes 1-methylinosine and participates in the formation of the Y-base (yW). Our results suggest that m(1)G37 existed in tRNA before the divergence of the three domains, and that a tRNA(m(1)G37)methyltrans ferase is part of the minimal set of gene products required for life.

  • 6.
    Björk, Glenn R
    et al.
    Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Wikström, P M
    Byström, Anders S
    Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Prevention of translational frameshifting by the modified nucleoside 1-methylguanosine1989In: Science, ISSN 0036-8075, E-ISSN 1095-9203, Vol. 244, no 4907, p. 986-989Article in journal (Refereed)
    Abstract [en]

    The methylated nucleoside 1-methylguanosine (m1G) is present next to the 3' end of the anticodon (position 37) in all transfer RNAs (tRNAs) that read codons starting with C except in those tRNAs that read CAN codons. All of the three proline tRNA species, which read CCN codons in Salmonella typhimurium, have been sequenced and shown to contain m1G in position 37. A mutant of S. typhimurium that lacks m1G in its tRNA when grown at temperatures above 37 degrees C, has now been isolated. The mutation (trmD3) responsible for this methylation deficiency is in the structural gene (trmD) for the tRNA(m1G37)methyltransferase. Therefore, the three proline tRNAs in the trmD3 mutant have an unmodified guanosine at position 37. Furthermore, the trmD3 mutation also causes at least one of the tRNAPro species to frequently shift frame when C's are present successively in the message. Thus, m1G appears to prevent frameshifting. The data from eubacteria apply to both eukaryotes and archaebacteria.

  • 7.
    Byström, Anders S
    et al.
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Björk, Glenn R
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Chromosomal location and cloning of the gene (trmD) responsible for the synthesis of tRNA (m1G) methyltransferase in Escherichia coli K-12.1982In: Molecular General Genetics, ISSN 0026-8925, E-ISSN 1432-1874, Vol. 188, no 3, p. 440-446Article in journal (Refereed)
    Abstract [en]

    The trmD gene, which governs the formation of 1-methyl-guanosine(m1G) in transfer ribonucleic acid (tRNA), has been located by phage P1 transduction at 56 min on the chromosomal map of Escherichia coli. Cotransduction to tyrA at 56 min is 80%. From the Clarke and Carbon collection a ColE1-tyrA+ hybrid plasmid was isolated, which carried the trmD+ gene and was shown to over-produce the tRNA(m1G)methyltransferase. By subcloning restriction enzyme fragments in vitro, the trmD+ gene was located to a 3.4 kb DNA fragment 6.5 kb clockwise from the tyrA+ gene. The mutation trmD1, which renders the tRNA(m1G)methyltransferase temperature-sensitive both in vivo and in vitro could be complemented by trmD+ plasmids. These results suggest that the gene trmD+ is the structural gene for the tRNA(m1G)methyltransferase (EC 2.1.1.3.1).

  • 8.
    Byström, Anders S
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Björk, Glenn R
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    The structural gene (trmD) for the tRNA(m1G)methyltransferase is part of a four polypeptide operon in Escherichia coli K-121982In: Molecular General Genetics, ISSN 0026-8925, E-ISSN 1432-1874, Vol. 188, no 3, p. 447-454Article in journal (Refereed)
    Abstract [en]

    The trmD gene, which is the structural gene for the tRNA(m1G)-methyltransferase, is shown to be part of a polycistronic operon. A 4.6 kb SalI-EcoRI chromosomal DNA fragment contains the trmD gene (Byström and Björk 1982). Subclonings, deletion mapping and Tn5 insertions into plasmid pBY03 have established the gene organization of the trmD area on the Escherichia coli chromosome. The different plasmid derivatives were analysed for expression of protein products using the minicell system. Such analyses established the organisation of genes encoding six polypeptides to be SalI1-48 K-13 K-25 K-31 K-15 K-16 K-EcoRI1. The 31 K polypeptide was shown to be the tRNA(m1G)methyltransferase. The trmD operon encodes for four polypeptides; 13 K-25 K-31 K(trmD)-15 K and the direction of transcription is from 13 K (promoter proximal) to 15 K (promoter distal). However, there might be a weak internal promoter between the trmD gene and the gene encoding the 15 K product. The level of expression from this operon in the minicell system does not seem to follow normal polarity since we observed high expression of 13 K, 25 K, and 15 K products but low expression of the internal trmD gene.

  • 9.
    Byström, Anders S
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Hjalmarsson, Karin J
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Wikström, P Mikael
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Björk, Glenn R
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    The nucleotide sequence of an Escherichia coli operon containing genes for the tRNA(m1G)methyltransferase, the ribosomal proteins S16 and L19 and a 21-K polypeptide1983In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 2, no 6, p. 899-905Article in journal (Refereed)
    Abstract [en]

    The nucleotide sequence of a 4.6-kb SalI-EcoRI DNA fragment including the trmD operon, located at min 56 on the Escherichia coli K-12 chromosome, has been determined. The trmD operon encodes four polypeptides: ribosomal protein S16 (rpsP), 21-K polypeptide (unknown function), tRNA-(m1G)methyltransferase (trmD) and ribosomal protein L19 (rplS), in that order. In addition, the 4.6-kb DNA fragment encodes a 48-K and a 16-K polypeptide of unknown functions which are not part of the trmD operon. The mol. wt. of tRNA(m1G)methyltransferase determined from the DNA sequence is 28 424. The probable locations of promoter and terminator of the trmD operon are suggested. The translational start of the trmD gene was deduced from the known NH2-terminal amino acid sequence of the purified enzyme. The intercistronic regions in the operon vary from 9 to 40 nucleotides, supporting the earlier conclusion that the four genes are co-transcribed, starting at the major promoter in front of the rpsP gene. Since it is known that ribosomal proteins are present at 8000 molecules/genome and the tRNA-(m1G)methyltransferase at only approximately 80 molecules/genome in a glucose minimal culture, some powerful regulatory device must exist in this operon to maintain this non-coordinate expression. The codon usage of the two ribosomal protein genes is similar to that of other ribosomal protein genes, i.e., high preference for the most abundant tRNA isoaccepting species. The trmD gene has a codon usage typical for a protein made in low amount in accordance with the low number of tRNA-(m1G)methyltransferase molecules found in the cell.

  • 10.
    Byström, Anders S
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    von Gabain, A
    Björk, Glenn R
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Differentially expressed trmD ribosomal protein operon of Escherichia coli is transcribed as a single polycistronic mRNA species1989In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 208, no 4, p. 575-586Article in journal (Refereed)
    Abstract [en]

    The trmD operon is a four-cistron operon in which the first and fourth genes encode ribosomal proteins S16 (rpsP) and L19 (rplS), respectively. The second gene encodes a 21,000 Mr polypeptide of unknown function and the third gene (trmD) encodes the enzyme tRNA(m1G37)methyltransferase, which catalyzes the formation of 1-methylguanosine (m1G) next to the 3' end of the anticodon (position 37) of some tRNAs in Escherichia coli. Here we show under all regulatory conditions studied, transcription initiates at one unique site, and the entire operon is transcribed into one polycistronic mRNA. Between the promoter and the first gene, rpsP, an attenuator-like structure is found (delta G = -18 kcal; 1 cal = 4.184 J), followed by four uridine residues. This structure is functional in vitro, and terminates more than two-thirds of the transcripts. The different parts of the trmD operon mRNA decay at a uniform rate. The stability of the trmD mRNA is not reduced with decreasing growth rate, which is in contrast to what has been found for other ribosomal protein mRNAs. Furthermore, earlier experiments have shown the existence of differential expression as well as non-co-ordinate regulation within the operon. Our results are consistent with the regulation of the trmD operon being due to some mechanism(s) operating at the post-transcriptional level, and do not involve differential degradation of different mRNA segments, internal promoters or internal terminators.

  • 11.
    Chen, Peng
    et al.
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Crain, Pamela F
    Näsvall, Joakim
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Pomerantz, Steven C
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Björk, Glenn
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    A "gain of function" mutation in a protein mediates production of novel modified nucleosides.2005In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 24, no 10, p. 1842-1851Article in journal (Refereed)
    Abstract [en]

    The mutation sufY204 mediates suppression of a +1 frameshift mutation in the histidine operon of Salmonella enterica serovar Typhimurium and synthesis of two novel modified nucleosides in tRNA. The sufY204 mutation, which results in an amino-acid substitution in a protein, is, surprisingly, dominant over its wild-type allele and thus it is a "gain of function" mutation. One of the new nucleosides is 5-methylaminomethyl-2-thiouridine (mnm(5)s(2)U34) modified by addition of a C(10)H(17) side chain of unknown structure. Increased amounts of both nucleosides in tRNA are correlated to gene dosage of the sufY204 allele, to an increased efficiency of frameshift suppression, and to a decreased amount of the wobble nucleoside mnm(5)s(2)U34 in tRNA. Purified tRNA(Gln)(cmnm(5)s(2)UUG) in the mutant strain contains a modified nucleoside similar to the novel nucleosides and the level of aminoacylation of tRNA(Gln)(cmnm(5)s(2)UUG) was reduced to 26% compared to that found in the wild type (86%). The results are discussed in relation to the mechanism of reading frame maintenance and the evolution of modified nucleosides in tRNA.

  • 12.
    Chen, Peng
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Qian, Qiang
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Zhao, Shaoping
    Isaksson, Leif A.
    Björk, Glenn R
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    A cytosolic tRNA with an unmodified adenosine in the wobble position reads a codon ending with the non-complementary nucleoside cytidine2002In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 317, no 4, p. 481-492Article in journal (Refereed)
    Abstract [en]

    Out of more than 500 sequenced cytosolic tRNAs, there is only one with an unmodified adenosine in the wobble position (position 34). The reason for this rare occurrence of A34 is that it is mostly deaminated to inosine-34 (I34). I34 is a common constituent in the wobble position of tRNAs and has a decoding capacity different from that of A34. We have isolated a mutant (proL207) of Salmonella typhimurium, in which the wobble nucleoside G34 has been replaced by an unmodified A in tRNA(Pro)(GGG), which is the only tRNA that normally reads the CCC codon. Thus, this mutant apparently has no tRNA that is considered cognate for the codon CCC. Despite this, the mutant grows normally. As expected, Pro-tRNA selection at the CCC codon in the A-site in a mutant deleted for the proL gene, which encodes the tRNA(Pro)(GGG), was severely reduced. However, in comparison this rate of selection was only slightly reduced in the proL207 mutant with its A34 containing tRNA(Pro)(AGG) suggesting that this tRNA reads CCC. Moreover, measurements of the interference by a tRNA residing in the P-site on the apparent termination efficiency at the A-site indicated that indeed the A34 containing tRNA reads the CCC codon. We conclude that A34 in a cytosolic tRNA is not detrimental to the cell and that the mutant tRNA(Pro)(AGG) is able to read the CCC codon like its wild-type counterpart tRNA(Pro)(GGG). We suggest that the decoding of the CCC codon by a 5'-AGG-3' anticodon occurs by a wobble base-pair between a protonated A34 and a C in the mRNA. Copyright 2002 Elsevier Science Ltd.

  • 13. Durand, Jérôme M B
    et al.
    Björk, Glenn R
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Metabolic control through ornithine and uracil of epithelial cell invasion by Shigella flexneri2009In: Microbiology, ISSN 1350-0872, E-ISSN 1465-2080, Vol. 155, no Pt 8, p. 2498-2508Article in journal (Refereed)
    Abstract [en]

    This paper shows that compounds in defined growth media strongly influence the expression of the effectors of virulence in the human invasive pathogen Shigella flexneri. Ornithine in conjunction with uracil reduces the haemolytic ability of wild-type cultures more than 20-fold and the expression of the type III secretion system more than 8-fold, as monitored by an mxiC : : lacZ transcriptional reporter. mxiC gene expression is further decreased by the presence of methionine or branched-chain amino acids (15-fold or 25-fold at least, respectively). Lysine and a few other aminated metabolites (cadaverine, homoserine and diaminopimelate) counteract the ornithine-mediated inhibition of haemolytic activity and of the expression of a transcriptional activator virF reporter. The complete abolition of invasion of HeLa cells by wild-type bacteria by ornithine, uracil, methionine or branched-chain amino acids establishes that these metabolites are powerful effectors of virulence. These findings provide a direct connection between metabolism and virulence in S. flexneri. The inhibitory potential exhibited by the nutritional environment is stronger than temperature, the classical environmental effector of virulence. The implications and practical application of this finding in prophylaxis and treatment of shigellosis are discussed.

  • 14. Gaur, Rahul
    et al.
    Björk, Glenn
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Tuck, Simon
    Umeå Centre for Molecular Pathogenesis (UCMP).
    Varshney, Umesh
    Diet-dependent depletion of queuosine in tRNAs in Caenorhabditis elegans does not lead to a developmental block.2007In: J Biosci, ISSN 0250-5991, Vol. 32, no 4, p. 747-54Article in journal (Refereed)
    Abstract [en]

    Queuosine (Q), a hypermodified nucleoside,occurs at the wobble position of transfer RNAs (tRNAs)with GUN anticodons. In eubacteria, absence of Q affects messenger RNA (mRNA) translation and reduces the virulence of certain pathogenic strains. In animal cells,changes in the abundance of Q have been shown to correlate with diverse phenomena including stress tolerance, cell proliferation and tumour growth but the function of Q in animals is poorly understood. Animals are thought to obtain Q (or its analogues) as a micronutrient from dietary sources such as gut micro flora. However,the difficulty of maintaining animals under bacteria-free conditions on Q-deficient diets has severely hampered the study of Q metabolism and function in animals. In this study,we show that as in higher animals, tRNAs in the nematode Caenorhabditis elegans are modified by Q and its sugar derivatives. When the worms were fed on Q-deficient Escherichia coli, Q modification was absent from the worm tRNAs suggesting that C.elegans lacks a de novo pathway of Q biosynthesis. The inherent advantages of C.elegans as a model organism, and the simplicity of conferring a Q-deficient phenotype on it make it an ideal system to investigate the function of Q modification in tRNA.

  • 15. Grosjean, Henri
    et al.
    Björk, Glenn
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Enzymatic conversion of cytidine to lysidine in anticodon of bacterial isoleucyl-tRNA--an alternative way of RNA editing2004In: TIBS -Trends in Biochemical Sciences. Regular ed., ISSN 0968-0004, E-ISSN 1362-4326, Vol. 29, no 4, p. 165-168Article in journal (Refereed)
    Abstract [en]

    In most organisms, the AUA triplet codes for isoleucine (Ile), whereas in a few organelles it codes for methionine (Met). In bacteria, this A-ending triplet is decoded by an unusual tRNA harboring a Met anticodon CAU, where cytidine at the wobble position 34 (C34) is posttranscriptionally modified to a 2-lysyl cytidine (lysidine), abbreviated as (k2C). Now, the bacterial gene tilS, which encodes the enzyme catalyzing the lysylation of C34 in the precursor tRNAIle(CAU), thereby leading to the formation of tRNAIle(k2CAU), has been identified. The formation of lysidine by this essential enzyme allows recognition of tRNAIle(k2CAU) by Ile-tRNA synthetase and switches the base pairing of the tRNA from AUG (Met) to AUA (Ile). This base change is reminiscent of C-to-U type of RNA editing of some mitochondrial tRNAs.

     

  • 16. Grosjean, Henri
    et al.
    de Crécy-Lagard, Valérie
    Björk, Glenn R
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Aminoacylation of the anticodon stem by a tRNA-synthetase paralog: relic of an ancient code?2004In: TIBS -Trends in Biochemical Sciences. Regular ed., ISSN 0968-0004, E-ISSN 1362-4326, Vol. 29, no 10, p. 519-522Article in journal (Refereed)
    Abstract [en]

    The activation and charging of amino acids onto the acceptor stems of their cognate tRNAs are the housekeeping functions of aminoacyl-tRNA synthetases. The availability of whole genome sequences has revealed the existence of synthetase-like proteins that have other functions linked to different aspects of cell metabolism and physiology. In eubacteria, a paralog of glutamyl tRNA synthetase, which lacks the tRNA-binding domain, was found to aminoacylate tRNA(Asp) not on the 3'-hydroxyl group of the acceptor stem but on a cyclopentene diol of the modified nucleoside queuosine present at the wobble position of anticodon loop. This modified nucleoside might be a relic of an ancient code.

  • 17. Gurvich, Olga L
    et al.
    Näsvall, S Joakim
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Baranov, Pavel V
    Björk, Glenn R
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Atkins, John F
    Two groups of phenylalanine biosynthetic operon leader peptides genes: a high level of apparently incidental frameshifting in decoding Escherichia coli pheL2011In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 39, no 8, p. 3079-3092Article in journal (Refereed)
    Abstract [en]

    The bacterial pheL gene encodes the leader peptide for the phenylalanine biosynthetic operon. Translation of pheL mRNA controls transcription attenuation and, consequently, expression of the downstream pheA gene. Fifty-three unique pheL genes have been identified in sequenced genomes of the gamma subdivision. There are two groups of pheL genes, both of which are short and contain a run(s) of phenylalanine codons at an internal position. One group is somewhat diverse and features different termination and 5’-flanking codons. The other group, mostly restricted to Enterobacteria and including Escherichia coli pheL, has a conserved nucleotide sequence that ends with UUC_CCC_UGA. When these three codons in E. coli pheL mRNA are in the ribosomal E-, P- and A-sites, there is an unusually high level, 15%, of +1 ribosomal frameshifting due to features of the nascent peptide sequence that include the penultimate phenylalanine. This level increases to 60% with a natural, heterologous, nascent peptide stimulator. Nevertheless, studies with different tRNA(Pro) mutants in Salmonella enterica suggest that frameshifting at the end of pheL does not influence expression of the downstream pheA. This finding of incidental, rather than utilized, frameshifting is cautionary for other studies of programmed frameshifting.

  • 18.
    Hjalmarsson, Karin J.
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Byström, Anders S
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Björk, Glenn R
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Purification and characterization of transfer RNA (guanine-1)methyltransferase from Escherichia coli1983In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 258, no 2, p. 1343-1351Article in journal (Refereed)
    Abstract [en]

    The tRNA modifying enzyme, tRNA (guanine-1)methyltransferase has been purified to near homogeneity from an overproducing Escherichia coli strain harboring a multicopy plasmid carrying the structural gene of the enzyme. The preparation gives a single major band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme is probably a single polypeptide chain of molecular weight 32,000. The amino acid composition is presented and the NH2-terminal amino acid sequence was established to be H2N-Met-Trp-Ile-Gly-Ile-Ile-Ser-Leu-Phe-Pro. The enzyme has a pI of 5.2. The tRNA (guanine-1)-methyltransferase has a pH optimum of 8.0-8.5, an apparent Km of 5 microM for S-adenosylmethionine. S-adenosylhomocysteine is a competitive inhibitor for the enzyme with an apparent Ki of 6 microM. Spermidine or putrescine are not required for activity, but they stimulate the rate of methylation 1.2-fold with optima at 2 and 6 mM, respectively. Ammonium ion is not required and is inhibitory at concentrations above 0.15 M. Magnesium ion inhibited the activity at a concentration as low as 2 mM. Sodium and potassium ions were inhibitory at concentrations above 0.1 M. The molecular activity of tRNA (guanine-1)-methyltransferase was calculated to 10.0 min-1. It was estimated that the enzyme is present at 80 molecules/genome in cells growing with a specific growth rate of 1.0.

  • 19.
    Johansson, Marcus J O
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Esberg, Anders
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Huang, Bo
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Björk, Glenn R
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Byström, Anders S
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Eukaryotic wobble uridine modifications promote a functionally redundant decoding system.2008In: Molecular and Cellular Biology, ISSN 0270-7306, E-ISSN 1098-5549, Vol. 28, no 10, p. 3301-3312Article in journal (Refereed)
    Abstract [en]

    The translational decoding properties of tRNAs are modulated by naturally occurring modifications of their nucleosides. Uridines located at the wobble position (nucleoside 34 [U34]) in eukaryotic cytoplasmic tRNAs often harbor a 5-methoxycarbonylmethyl (mcm(5)) or a 5-carbamoylmethyl (ncm(5)) side chain and sometimes an additional 2-thio (s2) or 2'-O-methyl group. Although a variety of models explaining the role of these modifications have been put forth, their in vivo functions have not been defined. In this study, we utilized recently characterized modification-deficient Saccharomyces cerevisiae cells to test the wobble rules in vivo. We show that mcm5 and ncm5 side chains promote decoding of G-ending codons and that concurrent mcm5 and s2 groups improve reading of both A- and G-ending codons. Moreover, the observation that the mcm5U34- and some ncm5U34-containing tRNAs efficiently read G-ending codons challenges the notion that eukaryotes do not use U-G wobbling.

  • 20.
    Jäger, Gunilla
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Chen, Peng
    Björk, Glenn R.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Transfer RNA Bound to MnmH Protein Is Enriched with Geranylated tRNA - A Possible Intermediate in Its Selenation?2016In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 11, no 4, article id e0153488Article in journal (Refereed)
    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.

  • 21.
    Jäger, Gunilla
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Leipuviene, Ramune
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Pollard, Michael
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Qian, Qiang
    Björk, Glenn
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    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.2004In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 186, no 3, p. 750-757Article in journal (Refereed)
    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.

  • 22.
    Jäger, Gunilla
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Nilsson, Kristina
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Björk, Glenn R.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    The Phenotype of Many Independently Isolated+1 Frameshift Suppressor Mutants Supports a Pivotal Role of the P-Site in Reading Frame Maintenance2013In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 4, p. e60246-Article in journal (Refereed)
    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.

  • 23. Kitamura, Aya
    et al.
    Nishimoto, Madoka
    Sengoku, Toru
    Shibata, Rie
    Jäger, Gunilla
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Björk, Glenn R.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    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)2012In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 287, no 52, p. 43950-43960Article in journal (Refereed)
    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.

  • 24.
    Leipuviene, Ramune
    et al.
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Björk, Glenn
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    A reduced level of charged tRNAArgmnm5UCU triggers the wild-type peptidyl-tRNA to frameshift.2005In: RNA, ISSN 1355-8382, Vol. 11, no 5, p. 796-807Article in journal (Refereed)
    Abstract [en]

    Frameshift mutations can be suppressed by a variety of differently acting external suppressors. The +1 frameshift mutation hisC3072, which has an extra G in a run of Gs, is corrected by the external suppressor mutation sufF44. We have shown that sufF44 and five additional allelic suppressor mutations are located in the gene argU coding for the minor tRNAArgmnm5UCU and alter the secondary and/or tertiary structure of this tRNA. The C61U, G53A, and C32U mutations influence the stability, whereas the C56U, C61U, G53A, and G39A mutations decrease the arginylation of tRNAArgmnm5UCU. The T-10C mutant has a base substitution in the -10 consensus sequence of the argU promoter that reduces threefold the synthesis of tRNAArgmnm5UCU . The lower amount of tRNAArgmnm5UCU or impaired arginylation, either independently or in conjunction, results in inefficient reading of the cognate AGA codon that, in turn, induces frameshifts. According to the sequence of the peptide produced from the suppressed -GGG-GAA-AGA- frameshift site, the frameshifting tRNA in the argU mutants is tRNAGlumnm5s2UUC, which decodes the GAA codon located upstream of the AGA arginine codon, and not the mutated tRNAArgmnm5UCU. We propose that an inefficient decoding of the AGA codon by a defective tRNAArgmnm5UCU stalls the ribosome at the A-site codon allowing the wild-type form of peptidyl-tRNAGlumnm5s2UUC to slip forward 1 nucleotide and thereby re-establish the ribosome in the 0-frame. Similar frame-shifting events could be the main cause of various phenotypes associated with environmental or genetically induced changes in the levels of aminoacylated tRNA.

  • 25.
    Leipuviene, Ramune
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Björk, Glenn R
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Alterations in the two globular domains or in the connecting alpha-helix of bacterial ribosomal protein L9 induces +1 frameshifts.2007In: J Bacteriol, ISSN 0021-9193, Vol. 189, no 19, p. 7024-31Article in journal (Refereed)
    Abstract [en]

    The ribosomal 50S subunit protein L9, encoded by the gene rplI, is an elongated protein with an alpha-helix connecting the N- and C-terminal globular domains. We isolated rplI mutants that suppress the +1 frameshift mutation hisC3072 in Salmonella enterica serovar Typhimurium. These mutants have amino acid substitutions in the N-terminal domain (G24D) or in the C-terminal domain (I94S, A102D, G126V, and F132S) of L9. In addition, different one-base deletions in rplI altered either the final portion of the C terminus or removed the C-terminal domain with or without the connecting alpha-helix. An alanine-to-proline substitution at position 59 (A59P), which breaks the alpha-helix between the globular domains, induced +1 frameshifting, suggesting that the geometrical relationship between the N and C domains is important to maintain the reading frame. Except for the alterations G126V in the C terminus and A59P in the connecting alpha-helix, our results confirm earlier results obtained by using the phage T4 gene 60-based system to monitor bypassing. The way rplI mutations suppress various frameshift mutations suggests that bypassing of many codons from several takeoff and landing sites occurred instead of a specific frameshift forward at overlapping codons.

  • 26.
    Leipuviene, Ramune
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Björk, Glenn R
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Ribosome slippage induced by mutations in the ribosomal protein L9Manuscript (Other academic)
  • 27.
    Leipuviene, Ramune
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Qian, Qiang
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Björk, Glenn
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Formation of thiolated nucleosides present in tRNA from Salmonella enterica serovar Typhimurium occurs in two principally distinct pathways.2004In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 186, no 3, p. 758-766Article in journal (Refereed)
    Abstract [en]

    tRNA from Salmonella enterica serovar Typhimurium contains five thiolated nucleosides, 2-thiocytidine (s(2)C), 4-thiouridine (s(4)U), 5-methylaminomethyl-2-thiouridine (mnm(5)s(2)U), 5-carboxymethylaminomethyl-2-thiouridine (cmnm(5)s(2)U), and N-6-(4-hydroxyisopentenyl)-2-methylthioadenosine (ms(2)io(6)A). The levels of all of them are significantly reduced in cells with a mutated iscS gene, which encodes the cysteine desulfurase IscS, a member of the ISC machinery that is responsible for [Fe-S] cluster formation in proteins. A mutant (iscU52) was isolated that carried an amino acid substitution (S107T) in the IscU protein, which functions as a major scaffold in the formation of [Fe-S] clusters. In contrast to the iscS mutant, the iscU52 mutant showed reduced levels of only two of the thiolated nucleosides, ms(2)io(6)A (10-fold) and s(2)C (more than 2-fold). Deletions of the iscU, hscA, or fdx genes from the isc operon lead to a similar tRNA thiolation pattern to that seen for the iscU52 mutant. Unexpectedly, deletion of the iscA gene, coding for an alternative scaffold protein for the [Fe-S] clusters, showed a novel tRNA thiolation pattern, where the synthesis of only one thiolated nucleoside, ms(2)io(6)A, was decreased twofold. Based on our results, we suggest two principal distinct routes for thiolation of tRNA: (i) a direct sulfur transfer from IscS to the tRNA modifying enzymes ThiI and MnmA, which form s(4)U and the s(2)U moiety of (c)mnm(5)s(2)U, respectively; and (ii) an involvement of [Fe-S] proteins (an unidentified enzyme in the synthesis of s(2)C and MiaB in the synthesis of ms(2)io(6)A) in the transfer of sulfur to the tRNA.

  • 28.
    Lundgren, Hans K
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Björk, Glenn R
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Structural alterations of the cysteine desulfurase IscS of Salmonella enterica serovar Typhimurium reveal substrate specificity of IscS in tRNA thiolation.2006In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 188, no 8, p. 3052-3062Article in journal (Refereed)
    Abstract [en]

    The cysteine desulfurase IscS in Salmonella enterica serovar Typhimurium is required for the formation of all four thiolated nucleosides in tRNA, which is thought to occur via two principally different biosynthetic pathways. The synthesis of 4-thiouridine (s(4)U) and 5-methylaminomethyl-2-thiouridine (mnm(5)s(2)U) occurs by a transfer of sulfur from IscS via various proteins to the target nucleoside in the tRNA, and no iron-sulfur cluster protein participates, whereas the synthesis of 2-thiocytidine (s(2)C) and N(6)-(4-hydroxyisopentenyl)-2-methylthioadenosine (ms(2)io(6)A) is dependent on iron-sulfur cluster proteins, whose formation and maintenance depend on IscS. Accordingly, inactivation of IscS should result in decreased synthesis of all thiolated nucleosides. We selected mutants defective either in the synthesis of a thiolated nucleoside (mnm(5)s(2)U) specific for the iron-sulfur protein-independent pathway or in the synthesis of a thiolated nucleoside (ms(2)io(6)A) specific for the iron-sulfur protein-dependent pathway. Although we found altered forms of IscS that influenced the synthesis of all thiolated nucleosides, consistent with the model, we also found mutants defective in subsets of thiolated nucleosides. Alterations in the C-terminal region of IscS reduced the level of only ms(2)io(6)A, suggesting that the synthesis of this nucleoside is especially sensitive to minor aberrations in iron-sulfur cluster transfer activity. Our results suggest that IscS has an intrinsic substrate specificity in how it mediates sulfur mobilization and/or iron-sulfur cluster formation and maintenance required for thiolation of tRNA.

  • 29. Martínez-Vicente, Marta
    et al.
    Yim, Lucía
    Villarroya, Magda
    Mellado, Mara
    Pérez-Payá, Enrique
    Björk, Glenn R
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Armengod, M-Eugenia
    Effects of mutagenesis in the switch I region and conserved arginines of Escherichia coli MnmE protein, a GTPase involved in tRNA modification2005In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 280, no 35, p. 30660-30670Article in journal (Refereed)
    Abstract [en]

    MnmE is an evolutionarily conserved, three domain GTPase involved in tRNA modification. In contrast to Ras proteins, MnmE exhibits a high intrinsic GTPase activity and requires GTP hydrolysis to be functionally active. Its G domain conserves the GTPase activity of the full protein, and thus, it should contain the catalytic residues responsible for this activity. In this work, mutational analysis of all conserved arginine residues of the MnmE G-domain indicates that MnmE, unlike other GTPases, does not use an arginine finger to drive catalysis. In addition, we show that residues in the G2 motif (249GTTRD253), which resides in the switch I region, are not important for GTP binding but play some role in stabilizing the transition state, specially Gly249 and Thr251. On the other hand, G2 mutations leading to a minor loss of the GTPase activity result in a non-functional MnmE protein. This indicates that GTP hydrolysis is a required but non-sufficient condition so that MnmE can mediate modification of tRNA. The conformational change of the switch I region associated with GTP hydrolysis seems to be crucial for the function of MnmE, and the invariant threonine (Thr251) of the G2 motif would be essential for such a change, because it cannot be substituted by serine. MnmE defects result in impaired growth, a condition that is exacerbated when defects in other genes involved in the decoding process are simultaneously present. This behavior is reminiscent to that found in yeast and stresses the importance of tRNA modification for gene expression.

  • 30. Mazauric, MH
    et al.
    Dirick, L
    Purushothaman, SK
    Björk, Glenn R
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Lapeyre, B
    Trm112p is a 15-kDa zinc finger protein essential for the activity of two tRNA and one protein methyltransferases in yeast2010In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 285, no 24, p. 18505-18515Article in journal (Refereed)
    Abstract [en]

    The degenerate base at position 34 of the tRNA anticodon is the target of numerous modification enzymes. In Saccharomyces cerevisiae, five tRNAs exhibit a complex modification of uridine 34 (mcm(5)U(34) and mcm(5)s(2)U(34)), the formation of which requires at least 25 different proteins. The addition of the last methyl group is catalyzed by the methyltransferase Trm9p. Trm9p interacts with Trm112p, a 15-kDa protein with a zinc finger domain. Trm112p is essential for the activity of Trm11p, another tRNA methyltransferase, and for Mtq2p, an enzyme that methylates the translation termination factor eRF1/Sup45. Here, we report that Trm112p is required in vivo for the formation of mcm(5)U(34) and mcm(5)s(2)U(34). When produced in Escherichia coli, Trm112p forms a complex with Trm9p, which renders the latter soluble. This recombinant complex catalyzes the formation of mcm(5)U(34) on tRNA in vitro but not mcm(5)s(2)U(34). An mtq2-0 trm9-0 strain exhibits a synthetic growth defect, thus revealing the existence of an unexpected link between tRNA anticodon modification and termination of translation. Trm112p is associated with other partners involved in ribosome biogenesis and chromatin remodeling, suggesting that it has additional roles in the cell.

  • 31. Moukadiri, Ismaïl
    et al.
    Garzón, M. -José
    Björk, Glenn R.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Armengod, M. -Eugenia
    The output of the tRNA modification pathways controlled by the Escherichia coli MnmEG and MnmC enzymes depends on the growth conditions and the tRNA species2014In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 42, no 4, p. 2602-2623Article in journal (Refereed)
    Abstract [en]

    In Escherichia coli, the MnmEG complex modifies transfer RNAs (tRNAs) decoding NNA/NNG codons. MnmEG catalyzes two different modification reactions, which add an aminomethyl (nm) or carboxymethylaminomethyl (cmnm) group to position 5 of the anticodon wobble uridine using ammonium or glycine, respectively. In tRNA(cmnm5s2UUG)(Gln) and tRNA(cmnm5UmAA)(Leu), however, cmnm(5) appears as the final modification, whereas in the remaining tRNAs, the MnmEG products are converted into 5-methylaminomethyl (mnm(5)) through the two-domain, bi-functional enzyme MnmC. MnmC(o) transforms cmnm(5) into nm(5), whereas MnmC(m) converts nm(5) into mnm(5), thus producing an atypical network of modification pathways. We investigate the activities and tRNA specificity of MnmEG and the MnmC domains, the ability of tRNAs to follow the ammonium or glycine pathway and the effect of mnmC mutations on growth. We demonstrate that the two MnmC domains function independently of each other and that tRNA(cmnm5s2UUG)(Gln) and tRNA(cmnm5UmAA)(Leu) are substrates for MnmC(m), but not MnmC(o). Synthesis of mnm(5)s(2) U by MnmEG-MnmC in vivo avoids build-up of intermediates in tRNA(mnm5s2UUU)(Lys). We also show that MnmEG can modify all the tRNAs via the ammonium pathway. Strikingly, the net output of the MnmEG pathways in vivo depends on growth conditions and tRNA species. Loss of any MnmC activity has a biological cost under specific conditions.

  • 32. Moukadiri, Ismaïl
    et al.
    Prado, Silvia
    Piera, Julio
    Velázquez-Campoy, Adrián
    Björk, Glenn R
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Armengod, M-Eugenia
    Evolutionarily conserved proteins MnmE and GidA catalyze the formation of two methyluridine derivatives at tRNA wobble positions2009In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 37, no 21, p. 7177-7193Article in journal (Refereed)
    Abstract [en]

    The wobble uridine of certain bacterial and mitochondrial tRNAs is modified, at position 5, through an unknown reaction pathway that utilizes the evolutionarily conserved MnmE and GidA proteins. The resulting modification (a methyluridine derivative) plays a critical role in decoding NNG/A codons and reading frame maintenance during mRNA translation. The lack of this tRNA modification produces a pleiotropic phenotype in bacteria and has been associated with mitochondrial encephalomyopathies in humans. In this work, we use in vitro and in vivo approaches to characterize the enzymatic pathway controlled by the Escherichia coli MnmE*GidA complex. Surprisingly, this complex catalyzes two different GTP- and FAD-dependent reactions, which produce 5-aminomethyluridine and 5-carboxymethylamino-methyluridine using ammonium and glycine, respectively, as substrates. In both reactions, methylene-tetrahydrofolate is the most probable source to form the C5-methylene moiety, whereas NADH is dispensable in vitro unless FAD levels are limiting. Our results allow us to reformulate the bacterial MnmE*GidA dependent pathway and propose a novel mechanism for the modification reactions performed by the MnmE and GidA family proteins.

  • 33.
    Nilsson, Kristina
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Jäger, Gunilla
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Björk, Glenn R.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    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?2017In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 12, no 4, article id e0175092Article in journal (Refereed)
    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.

  • 34.
    Nilsson, Kristina
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Lundgren, Hans K.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Hagervall, Tord G.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Björk, Glenn R
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    The cysteine desulfurase IscS is required for synthesis of all five thiolated nucleosides present in tRNA from Salmonella enterica serovar typhimurium2002In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 184, no 24, p. 6830-6835Article in journal (Refereed)
  • 35.
    Näsvall, Joakim
    et al.
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Chen, Peng
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Björk, Glenn
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    The modified wobble nucleoside uridine-5-oxyacetic acid in tRNAPro(cmo5UGG) promotes reading of all four proline codons in vivo.2004In: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 10, no 10, p. 1662-1673Article in journal (Refereed)
    Abstract [en]

    In Salmonella enterica serovar Typhimurium five of the eight family codon boxes are decoded by a tRNA having the modified nucleoside uridine-5-oxyacetic acid (cmo5U) as a wobble nucleoside present in position 34 of the tRNA. In the proline family codon box, one (tRNAProcmo5UGG) of the three tRNAs that reads the four proline codons has cmo5U34. According to theoretical predictions and several results obtained in vitro, cmo5U34 should base pair with A, G, and U in the third position of the codon but not with C. To analyze the function of cmo5U34 in tRNAProcmo5UGG in vivo, we first identified two genes (cmoA and cmoB) involved in the synthesis of cmo5U34. The null mutation cmoB2 results in tRNA having 5-hydroxyuridine (ho5U34) instead of cmo5U34, whereas the null mutation cmoA1 results in the accumulation of 5-methoxyuridine (mo5U34) and ho5U34 in tRNA. The results suggest that the synthesis of cmo5U34 occurs as follows: U34 -->(?) ho5U -->(CmoB) mo5U -->(CmoA?) cmo5U. We introduced the cmoA1 or the cmoB2 null mutations into a strain that only had tRNAProcmo5UGG and thus lacked the other two proline-specific tRNAs normally present in the cell. From analysis of growth rates of various strains and of the frequency of +1 frameshifting at a CCC-U site we conclude: (1) unexpectedly, tRNAProcmo5UGG is able to read all four proline codons; (2) the presence of ho5U34 instead of cmo5U34 in this tRNA reduces the efficiency with which it reads all four codons; and (3) the fully modified nucleoside is especially important for reading proline codons ending with U or C. Copyright 2004 RNA Society

  • 36.
    Näsvall, Joakim S
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Nilsson, Kristina
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Björk, Glenn R
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    The ribosomal grip of the peptidyl-tRNA is critical for reading frame maintenance.2009In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 385, no 2, p. 350-367Article in journal (Refereed)
    Abstract [en]

    If a ribosome shifts to an alternative reading frame during translation, the information in the message is usually lost. We have selected mutants of Salmonella typhimurium with alterations in tRNA(cmo5UGG)(Pro) that cause increased frameshifting when present in the ribosomal P-site. In 108 such mutants, two parts of the tRNA molecule are altered: the anticodon stem and the D-arm, including its tertiary interactions with the variable arm. Some of these alterations in tRNA(cmo5UGG)(Pro) are in close proximity to ribosomal components in the P-site. The crystal structure of the 30S subunit suggests that the C-terminal end of ribosomal protein S9 contacts nucleotides 32-34 of peptidyl-tRNA. We have isolated mutants with defects in the C-terminus of S9 that induce +1 frameshifting. Combinations of changes in tRNA(cmo5UGG)(Pro) and S9 suggest that an interaction occurs between position 32 of the peptidyl-tRNA and the C-terminal end of S9. Together, our results suggest that the cause of frameshifting is an aberrant interaction between the peptidyl-tRNA and the P-site environment. We suggest that the "ribosomal grip" of the peptidyl-tRNA is pivotal for maintaining the reading frame.

  • 37.
    Näsvall, S. Joakim
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Chen, Peng
    Björk, Glenn R.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    The Wobble hypothesis revisited: Uridine-5-oxyacetic acid is critical for reading of G-ending codons2007In: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 13, no 12, p. 2151-2164Article in journal (Refereed)
    Abstract [en]

    According to Crick's wobble hypothesis, tRNAs with uridine at the wobble position (position 34) recognize A- and G-, but not U- or C-ending codons. However, U in the wobble position is almost always modified, and Salmonella enterica tRNAs containing the modified nucleoside uridine-5-oxyacetic acid (cmo5U34) at this position are predicted to recognize U- (but not C-) ending codons, in addition to A- and G-ending codons. We have constructed a set of S. enterica mutants with only the cmo5U-containing tRNA left to read all four codons in the proline, alanine, valine, and threonine family codon boxes. From the phenotypes of these mutants, we deduce that the proline, alanine, and valine tRNAs containing cmo5U read all four codons including the C-ending codons, while the corresponding threonine tRNA does not. A cmoB mutation, leading to cmo5U deficiency in tRNA, was introduced. Monitoring A-site selection rates in vivo revealed that the presence of cmo5U34 stimulated the reading of CCU and CCC (Pro), GCU (Ala), and GUC (Val) codons. Unexpectedly, cmo5U is critical for efficient decoding of G-ending Pro, Ala, and Val codons. Apparently, whereas G34 pairs with U in mRNA, the reverse pairing (U34-G) requires a modification of U34.

  • 38. Sørensen, Michael A
    et al.
    Elf, Johan
    Bouakaz, Elli
    Tenson, Tanel
    Sanyal, Suparna
    Björk, Glenn
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Ehrenberg, Måns
    Over expression of a tRNA(Leu) isoacceptor changes charging pattern of leucine tRNAs and reveals new codon reading.2005In: J Mol Biol, ISSN 0022-2836, Vol. 354, no 1, p. 16-24Article in journal (Refereed)
    Abstract [en]

    During mRNA translation, synonymous codons for one amino acid are often read by different isoaccepting tRNAs. The theory of selective tRNA charging predicts greatly varying percentages of aminoacylation among isoacceptors in cells starved for their common amino acid. It also predicts major changes in tRNA charging patterns upon concentration changes of single isoacceptors, which suggests a novel type of translational control of gene expression. We therefore tested the theory by measuring with Northern blots the charging of Leu-tRNAs in Escherichia coli under Leu limitation in response to over expression of tRNA(GAG)(Leu). As predicted, the charged level of tRNA(GAG)(Leu) increased and the charged levels of four other Leu isoacceptors decreased or remained unchanged, but the charged level of tRNA(UAG)(Leu) increased unexpectedly. To remove this apparent inconsistency between theory and experiment we postulated a previously unknown common codon for tRNA(GAG)(Leu) and tRNA(UAG)(Leu). Subsequently, we demonstrated that the tRNA(GAG)(Leu) codon CUU is, in fact, read also by tRNA(UAG)(Leu), due to a uridine-5-oxyacetic acid modification.

  • 39.
    Urbonavicius, Jaunius
    et al.
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Jäger, Gunilla
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Björk, Glenn R
    Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine).
    Amino acid residues of the Escherichia coli tRNA(m5U54)methyltransferase (TrmA) critical for stability, covalent binding of tRNA and enzymatic activity.2007In: Nucleic Acids Res, ISSN 1362-4962, Vol. 35, no 10, p. 3297-305Article in journal (Refereed)
    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.

  • 40.
    Wikström, P M
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Byström, Anders S
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Björk, Glenn R
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Non-autogenous control of ribosomal protein synthesis from the trmD operon in Escherichia coli1988In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 203, no 1, p. 141-152Article in journal (Refereed)
    Abstract [en]

    The trmD operon of Escherichia coli encodes the ribosomal proteins S16 and L19, the tRNA(m1G37)methyltransferase and a 21,000 Mr protein of unknown function. Here we demonstrate that, in contrast to the expression of other ribosomal protein operons, the amount of trmD operon mRNA and the rate of synthesis of the proteins encoded by the operon respond to increased gene dosage. The steady-state level of the mRNA was about 18 times higher, and the relative rate of synthesis of the ribosomal proteins S16 and L19, the tRNA(m1G37)methyltransferase and the 21,000 Mr protein was 15, 9, 25 and 23 times higher, respectively, in plasmid-containing cells than in plasmid-free cells. Overproduced tRNA(m1G37)methyltransferase and 21,000 Mr protein were as stable as E. coli total protein, whereas the two ribosomal proteins were degraded to a large extent. The steady-state amount of S16 and L19 in the plasmid-containing cells exceeded that in plasmid-free cells by threefold and twofold, respectively. No significant effect on the synthesis of the trmD operon proteins from the chromosomally located genes was observed when parts of the operon were expressed on different plasmids. Taken together, these results suggest that the expression of the trmD operon is not subject to transcriptional or translational feedback regulation, and demonstrate that not all ribosomal protein operons are regulated in the same manner. We propose that ribosomal protein operons that do not encode proteins that bind directly to rRNA are not under autogenous control. Metabolic regulation at the transcriptional level and protein degradation are plausible mechanisms for the control of expression of such operons.

  • 41. Yim, Lucía
    et al.
    Moukadiri, Ismaïl
    Björk, Glenn
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Armengod, M-Eugenia
    Further insights into the tRNA modification process controlled by proteins MnmE and GidA of Escherichia coli.2006In: Nucleic Acids Res, ISSN 1362-4962, Vol. 34, no 20, p. 5892-905Article in journal (Refereed)
    Abstract [en]

    In Escherichia coli, proteins GidA and MnmE are involved in the addition of the carboxymethylaminomethyl (cmnm) group onto uridine 34 (U34) of tRNAs decoding two-family box triplets. However, their precise role in the modification reaction remains undetermined. Here, we show that GidA is an FAD-binding protein and that mutagenesis of the N-terminal dinucleotide-binding motif of GidA, impairs capability of this protein to bind FAD and modify tRNA, resulting in defective cell growth. Thus, GidA may catalyse an FAD-dependent reaction that is required for production of cmnmU34. We also show that GidA and MnmE have identical cell location and that both proteins physically interact. Gel filtration and native PAGE experiments indicate that GidA, like MnmE, dimerizes and that GidA and MnmE directly assemble in an alpha2beta2 heterotetrameric complex. Interestingly, high-performance liquid chromatography (HPLC) analysis shows that identical levels of the same undermodified form of U34 are present in tRNA hydrolysates from loss-of-function gidA and mnmE mutants. Moreover, these mutants exhibit similar phenotypic traits. Altogether, these results do not support previous proposals that activity of MnmE precedes that of GidA; rather, our data suggest that MnmE and GidA form a functional complex in which both proteins are interdependent.

1 - 41 of 41
CiteExportLink to result list
Permanent link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf