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Function of wobble nucleoside modifications in tRNAs of Salmonella enterica Serovar Typhimurium
Umeå University, Faculty of Medicine, Molecular Biology.
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 [en]
Molecular biology, tRNA, wobble nucleoside, frameshifting, translation
Keyword [sv]
Molekylärbiologi
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-328ISBN: 91-7305-734-7 (print)OAI: oai:DiVA.org:umu-328DiVA: diva2:143112
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
List of papers
1. A cytosolic tRNA with an unmodified adenosine in the wobble position reads a codon ending with the non-complementary nucleoside cytidine
Open this publication in new window or tab >>A cytosolic tRNA with an unmodified adenosine in the wobble position reads a codon ending with the non-complementary nucleoside cytidine
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2002 (English)In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 317, no 4, 481-492 p.Article in journal (Refereed) Published
Abstract [en]

Out of more than 500 sequenced cytosolic tRNAs, there is only one with an unmodified adenosine in the wobble position (position 34). The reason for this rare occurrence of A34 is that it is mostly deaminated to inosine-34 (I34). I34 is a common constituent in the wobble position of tRNAs and has a decoding capacity different from that of A34. We have isolated a mutant (proL207) of Salmonella typhimurium, in which the wobble nucleoside G34 has been replaced by an unmodified A in tRNA(Pro)(GGG), which is the only tRNA that normally reads the CCC codon. Thus, this mutant apparently has no tRNA that is considered cognate for the codon CCC. Despite this, the mutant grows normally. As expected, Pro-tRNA selection at the CCC codon in the A-site in a mutant deleted for the proL gene, which encodes the tRNA(Pro)(GGG), was severely reduced. However, in comparison this rate of selection was only slightly reduced in the proL207 mutant with its A34 containing tRNA(Pro)(AGG) suggesting that this tRNA reads CCC. Moreover, measurements of the interference by a tRNA residing in the P-site on the apparent termination efficiency at the A-site indicated that indeed the A34 containing tRNA reads the CCC codon. We conclude that A34 in a cytosolic tRNA is not detrimental to the cell and that the mutant tRNA(Pro)(AGG) is able to read the CCC codon like its wild-type counterpart tRNA(Pro)(GGG). We suggest that the decoding of the CCC codon by a 5'-AGG-3' anticodon occurs by a wobble base-pair between a protonated A34 and a C in the mRNA. Copyright 2002 Elsevier Science Ltd.

Keyword
inosine, decoding, wobble, tRNA, modification
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-4131 (URN)10.1006/jmbi.2002.5435 (DOI)11955004 (PubMedID)
Available from: 2004-10-01 Created: 2004-10-01 Last updated: 2010-03-19Bibliographically approved
2. The modified wobble nucleoside uridine-5-oxyacetic acid in tRNAPro(cmo5UGG) promotes reading of all four proline codons in vivo.
Open this publication in new window or tab >>The modified wobble nucleoside uridine-5-oxyacetic acid in tRNAPro(cmo5UGG) promotes reading of all four proline codons in vivo.
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

Keyword
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:nbn:se:umu:diva-16726 (URN)10.1261/rna.7106404 (DOI)15383682 (PubMedID)
Available from: 2007-10-09 Created: 2007-10-09 Last updated: 2010-03-19Bibliographically approved
3. Uridine-5-oxyacetic acid (cmo5U) present in the wobble position of a subset of tRNAs in Salmonella enterica Serovar Typhimurium has a tRNA dependent influence on coding capacity and cell physiology
Open this publication in new window or tab >>Uridine-5-oxyacetic acid (cmo5U) present in the wobble position of a subset of tRNAs in Salmonella enterica Serovar Typhimurium has a tRNA dependent influence on coding capacity and cell physiology
Manuscript (Other academic)
Identifiers
urn:nbn:se:umu:diva-4133 (URN)
Available from: 2004-10-01 Created: 2004-10-01 Last updated: 2010-01-13Bibliographically approved
4. A "gain of function" mutation in a protein mediates production of novel modified nucleosides.
Open this publication in new window or tab >>A "gain of function" mutation in a protein mediates production of novel modified nucleosides.
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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.

Keyword
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:nbn:se:umu:diva-16721 (URN)10.1038/sj.emboj.7600666 (DOI)15861125 (PubMedID)
Available from: 2007-10-09 Created: 2007-10-09 Last updated: 2010-03-19Bibliographically approved

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