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

Abstract

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.

Structural rearrangements in mRNA upon its binding to human 80S ribosomes revealed by EPR spectroscopy

“…The spin label 3-carboxy-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-1-oxyl succinimidyl ester (NHS-M2) was prepared according to the procedures described in Ref. ( 26 ) and used without deuteration, similar to our previous work ( 24 ). Undecaribonucleotide UGUGUUCGACA, whose 5′-terminal uridine and 3′-terminal adenosine bore ethylene diamine residues introduced at the C5 and С8 atoms, respectively, was synthesized and purified according to ( 27 ).…”

“…Undecaribonucleotide UGUGUUCGACA, whose 5′-terminal uridine and 3′-terminal adenosine bore ethylene diamine residues introduced at the C5 and С8 atoms, respectively, was synthesized and purified according to ( 27 ). The MR, doubly spin labeled RNA derivative that bore spin labels at the 5′ and 3′-terminal nucleotide bases, was prepared from the above oligomer by its treatment with NHS-M2 and subsequently purified as described in ( 24 ). Concentrations of the spin labels in sample containing MR were examined by EPR.…”

“…MR or tRNAs were labelled at their 5′-termini by gamma- 32 P-ATP and polynucleotide kinase as described ( 24 ). Mixtures for experiments on the analysis of binding of labeled MR to 80S ribosomes contained in 7 μl of buffer A 5.2 pmol of 80S ribosomes, 25 pmol of tRNA Phe and 4–45 pmol of labeled MR; in parallel, experiments without tRNA Phe were carried out.…”

“…Electron paramagnetic resonance (EPR) spectroscopy is actively used in studies of structure, dynamics and conformational changes of nucleic acids and their complexes ( 7 – 23 ). Recently, using doubly spin-labeled short model ss-RNA, we have shown that the EPR nanometer-scale distance measurements can be successfully applied to study the state of mRNA in the human ribosome ( 24 ). Thus, we anticipate that the extension of this approach to model mRNA forming rather stable secondary structure would open up a nice possibility to monitor structural changes in such mRNA upon its binding to 80S ribosomes.…”

Complementary-addressed site-directed spin labeling of long natural RNAs

“…Electron paramagnetic resonance (EPR) spectroscopy is actively used in studies of structure, dynamics and conformational changes of nucleic acids and their complexes ( 1 – 28 ). In this regard, RNAs evoke enormous interest of researchers because these biopolymers are extremely structurally dynamic macromolecules able to generate a wide set of conformations ( 29 ) and to form a variety of complexes with proteins ( 30 ).…”