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Structural alterations of the cysteine desulfurase IscS of Salmonella enterica serovar Typhimurium reveal substrate specificity of IscS in tRNA thiolation.
Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). (Björk)
Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). (Björk)
2006 (English)In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 188, no 8, 3052-3062 p.Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
2006. Vol. 188, no 8, 3052-3062 p.
Keyword [en]
Amino Acid Substitution, Carbon-Sulfur Lyases/*chemistry/genetics/*metabolism, Cytidine/analogs & derivatives/metabolism, DNA Mutational Analysis, Iron/metabolism, Isopentenyladenosine/analogs & derivatives/metabolism, Models; Biological, Models; Molecular, Mutation, Protein Biosynthesis, Protein Structure; Tertiary, RNA; Bacterial/metabolism, RNA; Transfer/*metabolism, Salmonella typhimurium/*enzymology, Substrate Specificity, Sulfur/metabolism, Thiouridine/analogs & derivatives/metabolism
URN: urn:nbn:se:umu:diva-16719DOI: 10.1128/JB.188.8.3052-3062.2006PubMedID: 16585765OAI: diva2:156392
Available from: 2007-10-09 Created: 2007-10-09 Last updated: 2010-04-22Bibliographically approved
In thesis
1. Formation of Thiolated Nucleosides in tRNA in Salmonella enterica serovar typhimurium
Open this publication in new window or tab >>Formation of Thiolated Nucleosides in tRNA in Salmonella enterica serovar typhimurium
2006 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The presence and synthesis of transfer RNA (tRNA) is highly conserved in all organisms and a lot of genetic material is dedicated to its synthesis. tRNA contains a large number of modified nucleosides and several diverse functions have been found but much about their function is still unknown. By using a novel frameshifting system to select for tRNA modification mutants, new mutations were isolated and subsequently analyzed. This thesis examines the synthesis and function of a subset of tRNA modifications that have a sulfur (thio) -group as part of the modification. The isc operon encodes for proteins synthesizing iron sulfur centers ([Fe-S]) that are a part of the active site of many key enzymes in the cell and the thiolated nucleosides are dependant on a functional iron sulfur gene (iscS) for their synthesis. By studying thiolated tRNA it is not only possible to learn more about the synthesis of the modifications themselves, but also about the synthesis of [Fe-S] clusters. Based on an analysis of mutations in three of the isc operon genes (iscS, iscU, and iscA), a two-model pathway is proposed for the synthesis of Salmonella enterica Serovar Typhimurium thiolated tRNA modifications. The interactions of IscS with other proteins in the tRNA modification thiolation pathways suggest a more complex sulfur relay than had previously been envisioned. Some of the specificities and the effect of an iscA mutant on the levels of tRNA modifications lead to an examination of the role of IscA in [Fe-S] formation and its importance for tRNA modifications.

Place, publisher, year, edition, pages
Umeå: Molekylärbiologi (Teknisk-naturvetenskaplig fakultet), 2006. 47 p.
: tRNA, modified nucleosides, thiolation, sulfur metabolism, Salmonella
National Category
Biochemistry and Molecular Biology
urn:nbn:se:umu:diva-857 (URN)91-7264-122-3 (ISBN)
Public defence
2006-10-13, Major Groove, 6L, Umeå Universitet, Umeå, 09:00 (English)
Available from: 2006-09-07 Created: 2006-09-07 Last updated: 2009-10-12Bibliographically approved

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