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Codon-reading specificities of mitochondrial release factors and translation termination at non-standard stop codons
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
2013 (English)In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 4Article in journal (Refereed) Published
Abstract [en]

A key feature of mitochondrial translation is the reduced number of transfer RNAs and reassignment of codons. For human mitochondria, a major unresolved problem is how the set of stop codons are decoded by the release factors mtRF1a and mtRF1. Here we present three-dimensional structural models of human mtRF1a and mtRF1 based on their homology to bacterial RF1 in the codon recognition domain, and the strong conservation between mitochondrial and bacterial ribosomal RNA in the decoding region. Sequence changes in the less homologous mtRF1 appear to be correlated with specific features of the mitochondrial rRNA. Extensive computer simulations of the complexes with the ribosomal decoding site show that both mitochondrial factors have similar specificities and that neither reads the putative vertebrate stop codons AGA and AGG. Instead, we present a structural model for a mechanism by which the ICT1 protein causes termination by sensing the presence of these codons in the A-site of stalled ribosomes.

Place, publisher, year, edition, pages
2013. Vol. 4
National Category
Biological Sciences
Identifiers
URN: urn:nbn:se:uu:diva-217675DOI: 10.1038/ncomms3940ISI: 000329396700004OAI: oai:DiVA.org:uu-217675DiVA: diva2:693768
Available from: 2014-02-05 Created: 2014-02-04 Last updated: 2017-12-06Bibliographically approved
In thesis
1. Computational Studies of Protein Synthesis on the Ribosome and Ligand Binding to Riboswitches
Open this publication in new window or tab >>Computational Studies of Protein Synthesis on the Ribosome and Ligand Binding to Riboswitches
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The ribosome is a macromolecular machine that produces proteins in all kingdoms of life. The proteins, in turn, control the biochemical processes within the cell. It is thus of extreme importance that the machine that makes the proteins works with high precision. By using three dimensional structures of the ribosome and homology modelling, we have applied molecular dynamics simulations and free-energy calculations to study the codon specificity of protein synthesis in initiation and termination on an atomistic level. In addition, we have examined the binding of small molecules to riboswitches, which can change the expression of an mRNA.

The relative affinities on the ribosome between the eukaryotic initiator tRNA to the AUG start codon and six near-cognate codons were determined. The free-energy calculations show that the initiator tRNA has a strong preference for the start codon, but requires assistance from initiation factors 1 and 1A to uphold discrimination against near-cognate codons.

When instead a stop codon (UAA, UGA or UAG) is positioned in the ribosomal A-site, a release factor binds and terminates protein synthesis by hydrolyzing the nascent peptide chain. However, vertebrate mitochondria have been thought to have four stop codons, namely AGA and AGG in addition to the standard UAA and UAG codons. Furthermore, two release factors have been identified, mtRF1 and mtRF1a. Free-energy calculations were used to determine if any of these two factors could bind to the two non-standard stop codons, and thereby terminate protein synthesis. Our calculations showed that the mtRF’s have similar stop codon specificity as bacterial RF1 and that it is highly unlikely that the mtRF’s are responsible for terminating at the AGA and AGG stop codons.

The eukaryotic release factor 1, eRF1, on the other hand, can read all three stop codons singlehandedly. We show that eRF1 exerts a high discrimination against near-cognate codons, while having little preference for the different cognate stop codons. We also found an energetic mechanism for avoiding misreading of the UGG codon and could identify a conserved cluster of hydrophobic amino acids which prevents excessive solvent molecules to enter the codon binding site.

The linear interaction energy method was used to examine binding of small molecules to the purine riboswitch and the FEP method was employed to explicitly calculate the LIE b-parameters. We show that the purine riboswitches have a remarkably high degree of electrostatic preorganization for their cognate ligands which is fundamental for discriminating against different purine analogs.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2017. 64 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1549
Keyword
Binding free energy, Ribosome, Codon reading, Translation initiation, Translation termination, Mitochondrial translation, Release factor, Purine riboswitch, Molecular Dynamics, Free Energy Perturbation
National Category
Biochemistry and Molecular Biology Bioinformatics (Computational Biology)
Research subject
Biology with specialization in Molecular Biotechnology
Identifiers
urn:nbn:se:uu:diva-328583 (URN)978-91-513-0051-1 (ISBN)
Public defence
2017-10-13, B41 BMC, Husargatan 3, Uppsala, 13:15 (English)
Opponent
Supervisors
Available from: 2017-09-22 Created: 2017-08-27 Last updated: 2017-10-17

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Lind, ChristofferSund, JohanÅqvist, Johan

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