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A "gain of function" mutation in a protein mediates production of novel modified nucleosides.
Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology). (Björk)
Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology). (Björk)
Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
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2005 (English)In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 24, no 10, 1842-1851 p.Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
2005. Vol. 24, no 10, 1842-1851 p.
Keyword [en]
Amino Acid Substitution, Frameshift Mutation, Genes; Suppressor, Lac Operon/genetics, Nucleosides/*biosynthesis/chemistry, Operon, RNA; Transfer/chemistry/genetics, Salmonella typhimurium/genetics/metabolism, Selenium Compounds/metabolism, Spectrometry; Mass; Electrospray Ionization, Transfer RNA Aminoacylation/genetics/physiology
National Category
Cell and Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-16721DOI: 10.1038/sj.emboj.7600666PubMedID: 15861125OAI: oai:DiVA.org:umu-16721DiVA: diva2:156394
Available from: 2007-10-09 Created: 2007-10-09 Last updated: 2010-03-19Bibliographically approved
In thesis
1. Wobble modifications and other features in transfer RNA important for decoding and reading frame maintenance
Open this publication in new window or tab >>Wobble modifications and other features in transfer RNA important for decoding and reading frame maintenance
2007 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Transfer RNA (tRNA) is the adaptor molecule responsible for bringing the correct amino acid to the ribosome during protein synthesis. tRNA contains a number of modified nucleosides, which are derivatives of the four normal nucleosides. A great variety of modifications are found in the anticodon loop, especially at the first (wobble) position of the anticodon. According to Crick’s wobble hypothesis, a uridine at the wobble position of tRNA recognize codons ending with A and G. Uridine-5-oxyacetic acid (cmo5U34), found at the wobble position of six species of tRNA in Salmonella enterica, have been predicted to expand the codon recognition of uridine to include U-ending, but not C-ending codons.

To study the function of cmo5U34 we have identified two genes, cmoA and cmoB, which are required for the synthesis of cmo5U34 in tRNA. We have shown that the proline, alanine and valine tRNAs containing cmo5U34 are capable of reading codons ending with any of the four nucleotides, while the threonine tRNA is not, and the importance of having cmo5U is different for the different tRNAs. In addition, we found that cmo5U is important for efficient reading of G-ending codons, which is surprising considering the wobble hypothesis, which states that uridine should read G-ending codons.

The dominant +1 frameshift suppressor sufY suppresses the hisC3737 +1 frameshift mutation. We have demonstrated that sufY induces frameshifting at CCC-CAA (Pro-Gln), when tRNAPro[cmo5UGG] occupies the P-site. sufY mutants accumulate novel modified nucleosides at the wobble position of tRNAs that should normally have (c)mnm5s2U34. The presence of an extra sidechain (C10H17) on the wobble nucleoside of tRNAGln[(c)mnm5s2U] leads to slow decoding of CAA codons, inducing a translational pause that allows the P-site peptidyl-tRNAPro[cmo5UGG] to slip into the +1 frame.

We have characterized 108 independent frameshift suppressor mutants in the gene encoding tRNAPro[cmo5UGG]. The altered tRNAs are still able to read all four proline codons in the A-site, but induce frameshifts after translocation into the P-site. Some of the mutations are in regions of the tRNA that are involved in interactions with components of the P-site. We hypothesize that the ribosomal P-site keeps a “grip” of the peptidyl-tRNA to prevent loss of the reading frame.

Place, publisher, year, edition, pages
Umeå: Molekylärbiologi (Teknisk-naturvetenskaplig fakultet), 2007. 50 p.
Keyword
Transfer RNA, Modified nucleosides, Decoding, Reading frame maintenance
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-1399 (URN)978-91-7264-437-3 (ISBN)
Public defence
2007-11-22, Major Groove, 6L, Institutionen för Molekylärbiologi, Umeå Universitet, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2007-10-23 Created: 2007-10-23 Last updated: 2010-03-19Bibliographically approved
2. Function of wobble nucleoside modifications in tRNAs of Salmonella enterica Serovar Typhimurium
Open this publication in new window or tab >>Function of wobble nucleoside modifications in tRNAs of Salmonella enterica Serovar Typhimurium
2004 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Transfer RNA from all organisms has modified nucleosides and position 34 (the wobble position) is one of the most extensively modified positions. Some wobble nucleoside modifications restrict codon choice (e.g. 5-methylaminomethyl-2-thiouridine, mnm5s2U) while some extend the decoding capacity (e.g. uridine-5-oxyacetic acid, cmo5U). In this thesis the influence of wobble nucleoside modification on cell physiology and translation efficiency and accuracy is described.

A mutant proL tRNA (proL207) was isolated that had an unmodified adenosine in the wobble position. Surprisingly, the proL207 mutant grows normally and is efficiently selected at the non-complementary CCC codon. The explanation of how an A34 containing tRNA can read CCC codon could be that a protonated A can form a base pair with C.

cmo5U (uridine-5-oxyacetic acid) is present in the wobble position of five tRNA species in S.enterica. Two genes (cmoA and cmoB) have been identified that are involved in the synthetic pathway of cmo5U. Mutants were constructed in alanine, valine, proline, and threonine codon boxes which left only a cmo5U containing tRNA present in the cell. The influence of cmo5U on growth or on A site selection rates of the ternary complex was found to be tRNA dependent.

During the study of the frameshift suppressor sufY of the hisC3737 frameshift mutation, a dominant mutation was found in YbbB protein, a selenouridine synthetase. The frameshifting occurs at CCC-CAA codon contexts and is specific for CAA codons, which are read by tRNAGlncmnm5s2UUG . The sufY204 mutation is a dominant mutation resulting in a change from Gly67 to Glu67 in the YbbB protein, and mediates the synthesis of several novel modified nucleosides/nucleotides (UKs) with unknown structure. The synthesis of these UKs is connected to the synthesis of cmnm5s2U34. The presence of UK on tRNAGlnU*UG reduced aminoacylation and therefore might account for the slow entry at CAA codons which could result in +1 frameshifting by P site tRNA. The selenourdine synthetase activity is not required for the synthesis of UKs. We hypothesize that an intrinsic activity that is low in the wild type protein has been elevated by the single amino acid substitution and results in the synthesis of UKs.

Place, publisher, year, edition, pages
Umeå: Molekylärbiologi (Teknisk-naturvetenskaplig fakultet), 2004. 52 p.
Keyword
Molecular biology, tRNA, wobble nucleoside, frameshifting, translation, Molekylärbiologi
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
Identifiers
urn:nbn:se:umu:diva-328 (URN)91-7305-734-7 (ISBN)
Public defence
2004-10-22, Major Groove, 6L, Dept. of Molecular Biology, Umeå University, Umeå, 10:00 (English)
Opponent
Supervisors
Available from: 2004-10-01 Created: 2004-10-01 Last updated: 2010-03-19Bibliographically approved

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Näsvall, JoakimBjörk, Glenn

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