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The Wobble hypothesis revisited: Uridine-5-oxyacetic acid is critical for reading of G-ending codons
Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
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.

Place, publisher, year, edition, pages
2007. Vol. 13, no 12, 2151-2164 p.
Keyword [en]
wobble hypothesis, translation, tRNA, modified nucleoside, decoding
Identifiers
URN: urn:nbn:se:umu:diva-2658DOI: 10.1261/rna.731007PubMedID: 17942742OAI: oai:DiVA.org:umu-2658DiVA: diva2:140888
Available from: 2007-10-23 Created: 2007-10-23 Last updated: 2017-12-14Bibliographically 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
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Available from: 2007-10-23 Created: 2007-10-23 Last updated: 2010-03-19Bibliographically approved

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