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The modified wobble nucleoside uridine-5-oxyacetic acid in tRNAPro(cmo5UGG) promotes reading of all four proline codons in vivo.
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). (Björk)
2004 (English)In: RNA: A publication of the RNA Society, ISSN 1355-8382, Vol. 10, no 10, 1662-1673 p.Article in journal (Refereed) Published
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

Place, publisher, year, edition, pages
2004. Vol. 10, no 10, 1662-1673 p.
Keyword [en]
Amino Acid Sequence, Base Sequence, Binding Sites, Codon/genetics, Frameshift Mutation, Lac Operon, Proline/*chemistry, Protein Biosynthesis, RNA; Bacterial/*chemistry/*genetics, RNA; Transfer; Pro/*chemistry/*genetics, Salmonella typhimurium/genetics, Uridine/*analogs & derivatives/*chemistry
National Category
Cell and Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-16726DOI: 10.1261/rna.7106404PubMedID: 15383682OAI: oai:DiVA.org:umu-16726DiVA: diva2:156399
Available from: 2007-10-09 Created: 2007-10-09 Last updated: 2010-03-19Bibliographically approved
In thesis
1. 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
2. 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

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