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Wobble modifications and other features in transfer RNA important for decoding and reading frame maintenance
Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
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 [en]
Transfer RNA, Modified nucleosides, Decoding, Reading frame maintenance
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-1399ISBN: 978-91-7264-437-3 (print)OAI: oai:DiVA.org:umu-1399DiVA: diva2:140891
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
List of papers
1. 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
2. The Wobble hypothesis revisited: Uridine-5-oxyacetic acid is critical for reading of G-ending codons
Open this publication in new window or tab >>The Wobble hypothesis revisited: Uridine-5-oxyacetic acid is critical for reading of G-ending codons
2007 (English)In: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 13, no 12, 2151-2164 p.Article in journal (Refereed) Published
Abstract [en]

According to Crick's wobble hypothesis, tRNAs with uridine at the wobble position (position 34) recognize A- and G-, but not U- or C-ending codons. However, U in the wobble position is almost always modified, and Salmonella enterica tRNAs containing the modified nucleoside uridine-5-oxyacetic acid (cmo5U34) at this position are predicted to recognize U- (but not C-) ending codons, in addition to A- and G-ending codons. We have constructed a set of S. enterica mutants with only the cmo5U-containing tRNA left to read all four codons in the proline, alanine, valine, and threonine family codon boxes. From the phenotypes of these mutants, we deduce that the proline, alanine, and valine tRNAs containing cmo5U read all four codons including the C-ending codons, while the corresponding threonine tRNA does not. A cmoB mutation, leading to cmo5U deficiency in tRNA, was introduced. Monitoring A-site selection rates in vivo revealed that the presence of cmo5U34 stimulated the reading of CCU and CCC (Pro), GCU (Ala), and GUC (Val) codons. Unexpectedly, cmo5U is critical for efficient decoding of G-ending Pro, Ala, and Val codons. Apparently, whereas G34 pairs with U in mRNA, the reverse pairing (U34-G) requires a modification of U34.

Keyword
wobble hypothesis, translation, tRNA, modified nucleoside, decoding
Identifiers
urn:nbn:se:umu:diva-2658 (URN)10.1261/rna.731007 (DOI)17942742 (PubMedID)
Available from: 2007-10-23 Created: 2007-10-23 Last updated: 2010-05-06Bibliographically approved
3. 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.
Show others...
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
4. The “ribosomal grip” of the peptidyl-tRNA is critical for reading frame maintenance?
Open this publication in new window or tab >>The “ribosomal grip” of the peptidyl-tRNA is critical for reading frame maintenance?
Manuscript (Other academic)
Identifiers
urn:nbn:se:umu:diva-2660 (URN)
Available from: 2007-10-23 Created: 2007-10-23 Last updated: 2010-01-13Bibliographically approved

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