umu.sePublications
Change search
CiteExportLink to record
Permanent link

Direct 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
Frameshifting as a tool in analysis of transfer RNA modification and translation
Umeå University, Faculty of Medicine, Molecular Biology.
2004 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Studies of ribosomal reading frame maintenance are often based on frameshift mutation suppression experiments. In this thesis, suppression of a frameshift mutation in Salmonella enterica serovar Typhimurium by a tRNA and a ribosomal protein are described. The +1 frameshift mutation hisC3072 (that contains an extra G in a run of Gs) is corrected by mutations in the argU gene coding for the minor tRNAArgmnm5UCU. The altered tRNAArgmnm5UCU has a decreased stability and reduced aminoacylation due to changed secondary and/or tertiary structure. Protein sequencing revealed that during the translation of the GAA-AGA frameshifting site the altered tRNAArgmnm5UCU reads the AGA codon inefficiently. This induces a ribosomal pause, allowing the tRNAGlumnm5s2UUC residing in the ribosomal P-site to slip forward one nucleotide. The same frameshift mutation (hisC3072) was also suppressed by defects in the large ribosomal subunit protein L9. Single base substitutions, truncations, and absence of this protein induced ribosome slippage. Mutated ribosome could shift to the overlapping codon in the +1 frame, or bypass to a codon further downstream in the +1 frame. The signal for stimulation of slippage and function of L9 needs to be investigated.

During the search for suppressors of the hisD3749 frameshift mutation, a spontaneous mutant was isolated in the iscU gene that contained greatly decreased levels of the thiolated tRNA modifications ms2io6A and s2C. The iscU gene belongs to the iscR-iscSUA-hscBA-fdx operon coding for proteins involved in the assembly of [Fe-S] clusters. As has been shown earlier, IscS influences the synthesis of all thiolated nucleosides in tRNA by mobilizing sulfur from cysteine. In this thesis, it is demonstrated that IscU, HscA, and Fdx proteins are required for the synthesis of the tRNA modifications ms2io6A and s2C but are dispensable for the synthesis of s4U and (c)mnm5s2U. Based on these results it is proposed that two distinct pathways exist in the formation of thiolated nucleosides in tRNA: one is an [Fe-S] cluster-dependent pathway for the synthesis of ms2io6A and s2C and the other is an [Fe-S] cluster-independent pathway for the synthesis of s4U and (c)mnm5s2U. MiaB is a [Fe-S] protein required for the introduction of sulfur in ms2io6A. TtcA is proposed to be involved in the synthesis of s2C. This protein contains a CXXC conserved motif essential for cytidine thiolation that, together with an additional CXXC motif in the C-terminus may serve as an [Fe-S] cluster ligation site.

Place, publisher, year, edition, pages
Umeå: Molekylärbiologi , 2004. , 57 p.
Keyword [en]
Molecular biology, minor tRNA, frameshifting, suppression, L9, hopping, tRNA thiolation, [Fe-S] cluster, [Fe-S] protein, TtcA
Keyword [sv]
Molekylärbiologi
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-302ISBN: 91-7305-709-6 (print)OAI: oai:DiVA.org:umu-302DiVA: diva2:143004
Public defence
2004-09-23, Major Groove, 6L, Umeå University, Dept. of Molecular Biology, SE-901 87 Umeå, Sweden, Umeå, 09:00
Opponent
Supervisors
Available from: 2004-09-01 Created: 2004-09-01 Last updated: 2010-08-04Bibliographically approved
List of papers
1. Decreased stability and reduced aminoacylation of a minor arginine tRNA mediates ribosomal frameshifting
Open this publication in new window or tab >>Decreased stability and reduced aminoacylation of a minor arginine tRNA mediates ribosomal frameshifting
Manuscript (Other academic)
Identifiers
urn:nbn:se:umu:diva-4047 (URN)
Available from: 2004-09-01 Created: 2004-09-01 Last updated: 2010-01-13Bibliographically approved
2. Ribosome slippage induced by mutations in the ribosomal protein L9
Open this publication in new window or tab >>Ribosome slippage induced by mutations in the ribosomal protein L9
Manuscript (Other academic)
Identifiers
urn:nbn:se:umu:diva-4048 (URN)
Available from: 2004-09-01 Created: 2004-09-01 Last updated: 2010-01-13Bibliographically approved
3. Formation of thiolated nucleosides present in tRNA from Salmonella enterica serovar Typhimurium occurs in two principally distinct pathways.
Open this publication in new window or tab >>Formation of thiolated nucleosides present in tRNA from Salmonella enterica serovar Typhimurium occurs in two principally distinct pathways.
2004 (English)In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 186, no 3, 758-766 p.Article in journal (Refereed) Published
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.

Keyword
Escherichia coli Proteins, HSP70 Heat-Shock Proteins/physiology, Iron-Sulfur Proteins/metabolism, Mutation, Nucleosides/*metabolism, Operon, RNA; Bacterial/*metabolism, RNA; Transfer/*metabolism, Salmonella typhimurium/*genetics/growth & development, Sulfhydryl Compounds/*metabolism, Thiouridine/metabolism
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:umu:diva-16737 (URN)10.1128/JB.186.3.758-766.2004 (DOI)14729702 (PubMedID)
Available from: 2007-10-09 Created: 2007-10-09 Last updated: 2017-12-14Bibliographically approved
4. 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.
Open this publication in new window or tab >>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.
Show others...
2004 (English)In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 186, no 3, 750-757 p.Article in journal (Refereed) Published
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.

Keyword
Amino Acid Motifs, Arginine/metabolism, Bacterial Proteins/*chemistry/physiology, Codon, Conserved Sequence, Cytidine/*metabolism, Oxidation-Reduction, Protein Biosynthesis, RNA; Bacterial/*metabolism, RNA; Transfer/*metabolism, Salmonella typhimurium/*chemistry/genetics, Sulfhydryl Compounds/*metabolism, Sulfur/metabolism
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:umu:diva-16727 (URN)10.1128/JB.186.3.750-757.2004 (DOI)14729701 (PubMedID)
Available from: 2007-10-09 Created: 2007-10-09 Last updated: 2017-12-14Bibliographically approved

Open Access in DiVA

fulltext(685 kB)486 downloads
File information
File name FULLTEXT01.pdfFile size 685 kBChecksum SHA-1
fcae8f6b5540b618a2959b186379a9273568b2b7438b90260d9354766ecbd2bcd6d70a92
Type fulltextMimetype application/pdf

By organisation
Molecular Biology
Biochemistry and Molecular Biology

Search outside of DiVA

GoogleGoogle Scholar
Total: 486 downloads
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

isbn
urn-nbn

Altmetric score

isbn
urn-nbn
Total: 342 hits
CiteExportLink to record
Permanent link

Direct 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