Doubly Spin-Labeled RNA as an EPR Reporter for Studying Multicomponent Supramolecular Assemblies

Doubly Spin-Labeled RNA as an EPR Reporter for Studying Multicomponent Supramolecular Assemblies Full article

Journal

Biophysical Journal
ISSN: 0006-3495

Output data

Year: 2015, Volume: 109, Number: 12, Pages: 2637-2643 Pages count : DOI: 10.1016/j.bpj.2015.10.042

Authors

Malygin Alexey A. ^1,4 , Graifer Dmitri M. ^1,4 , Meschaninova Maria I. ¹ , Venyaminova Aliya G. ¹ , Krumkacheva Olesya A. ^2,4 , Fedin Matvey V. ^2,4 , Karpova Galina G. ^1,4 , Bagryanskaya Elena G. ^3,4

Affiliations

1	(Данные Web of science) RAS, Inst Chem Biol & Fundamental Med, SB, Moscow, Russia
2	(Данные Web of science) RAS, Int Tomog Ctr, SB, Moscow, Russia
3	(Данные Web of science) RAS, NN Vorozhtsov Novosibirsk Inst Organ Chem, SB, Moscow, Russia
4	(Данные Web of science) Novosibirsk State Univ, Novosibirsk 630090, Russia

mRNAs are involved in complicated supramolecular complexes with human 40S and 80S ribosomes responsible for the protein synthesis. In this work, a derivative of nonaribonucleotide pUUCGUAAAA with nitroxide spin labels attached to the 5'-phosphate and to the C8 atom of the adenosine in sixth position (mRNA analog) was used for studying such complexes using double electron-electron resonance/pulsed electron-electron double resonance spectroscopy. The complexes were assembled with participation of tRNA(Phe), which targeted triplet UUC of the derivative to the ribosomal peptidyl site and predetermined location of the adjacent GUA triplet coding for Val at the aminoacyl (A) site. The interspin distances were measured between the two labels of mRNA analog attached to the first nucleotide of the peptidyl site bound codon and to the third nucleotide of the A site bound codon, in the absence/presence of second tRNA bound at the A site. The values of the obtained interspin distances agree with those calculated for available near-atomic structures of similar complexes of 40S and 80S ribosomes, showing that neither 60S subunit nor tRNA at the A site have a noticeable effect on arrangement of mRNA at the codon-anticodon interaction area. In addition, the shapes of distance distributions in four studied ribosomal complexes allowed conclusions on conformational flexibility of mRNA in these complexes. Overall, the results of this study are the first, to our knowledge, demonstration of double electron-electron resonance/pulsed electron-electron double resonance application for measurements of intramolecular distances in multicomponent supramolecular complexes involving intricate cellular machineries and for evaluating dynamic properties of ligands bound to these machineries.

Malygin A.A. , Graifer D.M. , Meschaninova M.I. , Venyaminova A.G. , Krumkacheva O.A. , Fedin M.V. , Karpova G.G. , Bagryanskaya E.G.
Doubly Spin-Labeled RNA as an EPR Reporter for Studying Multicomponent Supramolecular Assemblies

Biophysical Journal. 2015. V.109. N12. P.2637-2643. DOI: 10.1016/j.bpj.2015.10.042 WOS Scopus РИНЦ

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Dec 1, 2015

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