| 1. |
- Hägerdal, Hans, 1960-
(författare)
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Periphery and Bridgehead. : A Synthesis of West Balinese History
- 2002
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Ingår i: Indonesia and the Malay World. - Basingstoke : Carfax Publishing. - 1363-9811 .- 1469-8382. ; 30, s. 145-192
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Tidskriftsartikel (refereegranskat)abstract
- The article provides a historical syntesis of West Bali as a region, showing how this often neglected part of the island has served as bridgehead to Java/Central Indonesia in spite of its peripherical political position.
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| 2. |
- Alkemar, Gunnar, et al.
(författare)
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A possible tertiary rRNA interaction between expansion segments ES3 and ES6 in eukaryotic 40S ribosomal subunits
- 2003
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Ingår i: RNA. - : Cold Spring Harbor Laboratory. - 1355-8382 .- 1469-9001. ; 9:1, s. 20-24
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Tidskriftsartikel (refereegranskat)abstract
- Eukaryotic 16S-like ribosomal RNAs contain 12 so-called expansion segments, i.e., sequences not included in the RNA secondary structure core common to eubacteria, archaea, and eukarya. Two of these expansion segments, ES3 and ES6, are juxtaposed in the recent three-dimensional model of the eukaryotic 40S ribosomal subunit. We have analyzed ES3 and ES6 sequences from more than 2900 discrete eukaryotic species, for possible sequence complementarity between the two expansion segments. The data show that ES3 and ES6 could interact by forming a helix consisting of seven to nine contiguous base pairs in almost all analyzed species. We, therefore, suggest that ES3 and ES6 form a direct RNA-RNA contact in the ribosome.
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| 3. |
- Alkemar, Gunnar, et al.
(författare)
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Secondary structure of two regions in expansion segments ES3 and ES6 with the potential of forming a tertiary interaction in eukaryotic 40S ribosomal subunits
- 2004
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Ingår i: RNA. - : Cold Spring Harbor Laboratory. - 1355-8382 .- 1469-9001. ; 10:3, s. 403-411
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Tidskriftsartikel (refereegranskat)abstract
- The 18S rRNA of the small eukaryotic ribosomal subunit contains several expansion segments. Electron microscopy data indicate that two of the largest expansion segments are juxtaposed in intact 40S subunits, and data from phylogenetic sequence comparisons indicate that these two expansion segments contain complementary sequences that could form a direct tertiary interaction on the ribosome. We have investigated the secondary structure of the two expansion segments in the region around the putative tertiary interaction. Ribosomes from yeast, wheat, and mouse-three organisms representing separate eukaryotic kingdoms-were isolated, and the structure of ES3 and part of the ES6 region were analyzed using the single-strand-specific chemical reagents CMCT and DMS and the double-strand-specific ribonuclease V1. The modification patterns were analyzed by primer extension and gel electrophoresis on an ABI 377 automated DNA sequencer. The investigated sequences were relatively exposed to chemical and enzymatic modification. This is in line with their indicated location on the surface at the solvent side of the subunit. The complementary ES3 and ES6 sequences were clearly inaccessible to single-strand modification, but available for cleavage by double-strand-specific RNase V1. The results are compatible with a direct helical interaction between bases in ES3 and ES6. Almost identical results were obtained with ribosomes from the three organisms investigated.
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| 4. |
- Bratt, E, et al.
(författare)
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Coordination of editing and splicing of glutamate receptor pre-mRNA
- 2003
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Ingår i: RNA. - : Cold Spring Harbor Laboratory. - 1355-8382 .- 1469-9001. ; 9, s. 309-318
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Tidskriftsartikel (refereegranskat)abstract
- Adenosine deaminase that acts on RNA, ADAR, catalyzes the conversion of adenosine into inosine within double-stranded RNA. This type of editing has mainly been found in genes involved in neurotransmission. Site-specific A to I modifications often require intronic sequences to create the double-stranded structure necessary for editing. A system was developed to investigate if editing and splicing of pre-mRNA are coordinated. We have focused on a selectively edited site (R/G) in the glutamate receptor subunit B pre-mRNA. This editing site is situated in close proximity to a 5′ splice site. To ensure efficient splicing, the editing site, together with its natural 5′ splice site, was fused to a 3′ splice site of the major late transcript from adenovirus. In vitro, on a premade transcript, ADAR2 editing and splicing were found to interfere with each other. The stable stem-loop required for ADAR2 editing had a negative effect on in vitro splicing, possibly by sequestering the 5′ splice site. Further, RNA helicase A was shown to overcome the splicing inhibition caused by ADAR2. In vivo, allowing cotranscriptional processing, the same construct was found to efficiently edit and splice without interference, suggesting that the two RNA processing events are coordinated.
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| 5. |
- Johansson, Marcus J O, et al.
(författare)
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Dual function of the tRNA(m(5)U54)methyltransferase in tRNA maturation
- 2002
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Ingår i: RNA. - 1355-8382 .- 1469-9001. ; 8:3, s. 324-335
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Tidskriftsartikel (refereegranskat)abstract
- A 5-methyluridine (m(5)U) residue at position 54 is a conserved feature of bacterial and eukaryotic tRNAs. The methylation of U54 is catalyzed by the tRNA(m5U54)methyltransferase, which in Saccharomyces cerevisiae is encoded by the nonessential TRM2 gene. In this study, we identified four different strains with mutant forms of tRNA(Ser)CGA. The absence of the TRM2 gene in these strains decreased the stability of tRNA(Ser)CGA and induced lethality. Two alleles of TRM2 encoding catalytically inactive tRNA(m5U54)methyltransferases were able to stabilize tRNA(Ser)CGA in one of the mutants, revealing a role for the Trm2 protein per se in tRNA maturation. Other tRNA modification enzymes interacting with tRNA(Ser)CGA in the maturation process, such as Pus4p, Trm1 p, and Trm3p were essential or important for growth of the tRNA(Ser)CGA mutants. Moreover, Lhp1p, a protein binding RNA polymerase III transcripts, was required to stabilize the mutant tRNAs. Based on our results, we suggest that tRNA modification enzymes might have a role in tRNA maturation not necessarily linked to their known catalytic activity.
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| 6. |
- Klaue, Y, et al.
(författare)
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Biochemical analysis and scanning force microscopy reveal productive and non-productive ADAR2 binding to RNA substrates
- 2003
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Ingår i: RNA. - : Cold Spring Harbor Laboratory. - 1355-8382 .- 1469-9001. ; 9:7, s. 839-846
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Tidskriftsartikel (refereegranskat)abstract
- Scanning force microscopy (SFM) can be used to image biomolecules at high resolution. Here we demonstrate that single-molecule analysis by SFM complements biochemical data on RNA protein binding and can provide information that cannot be obtained by the usual biochemical methods. We have used this method to study the interaction between the RNA editing enzyme ADAR2 and RNA transcripts containing selective and nonselective editing sites. The natural selectively edited R/G site from glutamate receptor subunit B (GluR-B) was inserted into an RNA backbone molecule consisting of a completely double-stranded (ds) central part and incompletely paired ends derived from potato spindle tuber viroid (PSTVd). This molecule was efficiently edited at the R/G site, but promiscuous editing occurred at nonselective sites in the completely double-stranded region. The construct was also used to analyze binding of ADAR2 to wild-type and modified R/G editing sites in relation to binding at other nonselectively edited sites. Editing analysis together with SFM allow us to differentiate between binding and enzymatic activity. ADAR2 has been reported to have a general affinity to dsRNA. However, we show that there is a prominent bias for stable binding at sites selectively edited over other edited sites. On the other hand, promiscuous editing at nonselective sites apparently results from transient binding of the enzyme to the substrate. Furthermore, we find distinct sites with nonproductive binding of the enzyme.
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| 7. |
- Lövgren, Mattias, et al.
(författare)
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The PRC-barrel domain of the ribosome maturation protein RimM mediates binding to ribosomal protein S19 in the 30S ribosomal subunits
- 2004
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Ingår i: RNA. - : Cold Spring Harbor Laboratory Press (CSHL). - 1355-8382 .- 1469-9001. ; 10:11, s. 1798-1812
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Tidskriftsartikel (refereegranskat)abstract
- The RimM protein in Escherichia coli is associated with free 30S ribosomal subunits but not with 70S ribosomes. A DeltarimM mutant is defective in 30S maturation and accumulates 17S rRNA. To study the interaction of RimM with the 30S and its involvement in 30S maturation, RimM amino acid substitution mutants were constructed. A mutant RimM (RimM-YY-->AA), containing alanine substitutions for two adjacent tyrosines within the PRC beta-barrel domain, showed a reduced binding to 30S and an accumulation of 17S rRNA compared to wild-type RimM. The (RimM-YY-->AA) and DeltarimM mutants had significantly lower amounts of polysomes and also reduced levels of 30S relative to 50S compared to a wild-type strain. A mutation in rpsS, which encodes r-protein S19, suppressed the polysome- and 16S rRNA processing deficiencies of the RimM-YY-->AA but not that of the DeltarimM mutant. A mutation in rpsM, which encodes r-protein S13, suppressed the polysome deficiency of both rimM mutants. Suppressor mutations, found in either helices 31 or 33b of 16S rRNA, improved growth of both the RimM-YY-->AA and DeltarimM mutants. However, they suppressed the 16S rRNA processing deficiency of the RimM-YY-->AA mutant more efficiently than that of the DeltarimM mutant. Helices 31 and 33b are known to interact with S13 and S19, respectively, and S13 is known to interact with S19. A GST-RimM but not a GST-RimM(YY-->AA) protein bound strongly to S19 in 30S. Thus, RimM likely facilitates maturation of the region of the head of 30S that contains S13 and S19 as well as helices 31 and 33b.
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| 8. |
- Nordlund, Monica E, et al.
(författare)
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Identification of the TRM2 gene encoding the tRNA(m5U54)methyltransferase of Saccharomyces cerevisiae.
- 2000
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Ingår i: RNA. - 1355-8382 .- 1469-9001. ; 6:6, s. 844-60
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Tidskriftsartikel (refereegranskat)abstract
- The presence of 5-methyluridine (m5U) at position 54 is a ubiquitous feature of most bacterial and eukaryotic elongator tRNAs. In this study, we have identified and characterized the TRM2 gene that encodes the tRNA(m5U54)methyltransferase, responsible for the formation of this modified nucleoside in Saccharomyces cerevisiae. Transfer RNA isolated from TRM2-disrupted yeast strains does not contain the m5U54 nucleoside. Moreover, a glutathione S-transferase (GST) tagged recombinant, Trm2p, expressed in Escherichia coli displayed tRNA(m5U54)methyltransferase activity using as substrate tRNA isolated from a trm2 mutant strain, but not tRNA isolated from a TRM2 wild-type strain. In contrast to what is found for the tRNA(m5U54)methyltransferase encoding gene trmA+ in E. coli, the TRM2 gene is not essential for cell viability and a deletion strain shows no obvious phenotype. Surprisingly, we found that the TRM2 gene was previously identified as the RNC1/NUD1 gene, believed to encode the yNucR endo-exonuclease. The expression and activity of the yNucR endo-exonuclease is dependent on the RAD52 gene, and does not respond to increased gene dosage of the RNC1/NUD1 gene. In contrast, we find that the expression of a trm2-LacZ fusion and the activity of the tRNA(m5U54)methyltransferase is not regulated by the RAD52 gene and does respond on increased gene dosage of the TRM2 (RNC1/NUD1) gene. Furthermore, there was no nuclease activity associated with a GST-Trm2 recombinant protein. The purified yNucR endo-exonuclease has been reported to have an NH2-D-E-K-N-L motif, which is not found in the Trm2p. Therefore, we suggest that the yNucR endo-exonuclease is encoded by a gene other than TRM2.
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| 9. |
- Näsvall, Joakim, et al.
(författare)
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The modified wobble nucleoside uridine-5-oxyacetic acid in tRNAPro(cmo5UGG) promotes reading of all four proline codons in vivo.
- 2004
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Ingår i: RNA. - : Cold Spring Harbor Laboratory. - 1355-8382 .- 1469-9001. ; 10:10, s. 1662-1673
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Tidskriftsartikel (refereegranskat)abstract
- 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
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| 10. |
- Ren, Yan-Guo, et al.
(författare)
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Inhibition of Klenow DNA polymerase and poly(A)-specific ribonuclease by aminoglycosides
- 2002
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Ingår i: RNA. - 1355-8382 .- 1469-9001. ; 8:11, s. 1393-1400
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Tidskriftsartikel (refereegranskat)abstract
- Aminoglycosides are known to bind and perturb the function of catalytic RNA. Here we show that they also are potent inhibitors of protein-based catalysis using Escherichia coli Klenow polymerase (pol) and mammalian poly(A)-specific ribonuclease (PARN) as model enzymes. The inhibition was pH dependent and released in a competitive manner by Mg2+. Kinetic analysis showed that neomycin B behaved as a mixed noncompetitive inhibitor. Iron-mediated hydroxyl radical cleavage was used to show that neomycin B interfered with metal-ion binding in the active sites of both enzymes. Our analysis suggests a mechanism of inhibition where the aminoglycoside binds in the active site of the enzyme and thereby displaces catalytically important divalent metal ions. The potential causes of aminoglycoside toxicity and the usage of aminoglycosides to probe, characterize, and perturb metalloenzymes are discussed.
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