Russian Journal of Biological Physics and Chemisrty Russian Journal of Biological Physics and Chemisrty АКТУАЛЬНЫЕ ВОПРОСЫ БИОЛОГИЧЕСКОЙ ФИЗИКИ И ХИМИИ 2499-9962 54040 МОЛЕКУЛЯРНАЯ БИОФИЗИКА И ФИЗИКА БИОМОЛЕКУЛ MOLECULAR BIOPHYSICS AND PHYSICS OF BIOMOLECULES МОЛЕКУЛЯРНАЯ БИОФИЗИКА И ФИЗИКА БИОМОЛЕКУЛ STRUCTURAL STUDIES OF NUCLEOTIDE- AND RNA-BINDING PROPERTIES OF THE PROTEINS, WHICH ACT AS TRANSCRIPTIONAL REGULATORS СТРУКТУРНЫЕ ИССЛЕДОВАНИЯ НУКЛЕОТИД- И РНК-СВЯЗЫВАЮЩИХ СВОЙСТВ БЕЛКОВ- РЕГУЛЯТОРОВ ТРАНСКРИПЦИИ Леконцева Н В Lekontseva N V natalja-lekontseva@rambler.ru Немчинова М С Nemchinova M S Балобанов В А Balobanov V A Мурина В Н Murina V N Никулин А Д Nikulin A D ФГБУН Институт белка РАН ru Institute of protein research RAS ru ФГБУН Институт белка РАН ru Institute of protein research RAS ru ФГБУН Институт белка РАН ru Institute of protein research RAS ru ФГБУН Институт белка РАН ru Institute of protein research RAS ru ФГБУН Институт белка РАН ru Institute of protein research RAS ru 25 06 2016 25 06 2016 1 1 246 250 20 06 2016 20 06 2016 https://rusjbpc.ru/en/nauka/article/54040/view

При структурных исследованиях РНК-связывающих свойств белка Hfq было обнаружено, что связывание индивидуальных рибонуклеотидов с белком происходит аналогично связыванию соответствующих оснований нуклеотидов в составе комплексов белка с одноцепочечными РНК. На основании этого наблюдения мы предложили методику идентификации мест специфического узнавания оснований РНК на поверхности белка с помощью рентгеноструктурного анализа. Предложенный подход был протестирован на двух РНК- связывающих белках - CspB из Bacillus subtilis и Rop из Escherichia coli. Для оценки полученных результатов были рассчитаны константы диссоциации комплексов исследуемых белков с АМФ и ГМФ по изменению анизотропии флуоресценции меченых нуклеотидов АМФ-МАНТ и ГМФ-МАНТ.

Earlier we have shown that positions of ribonucleotides in the nucleotide-Hfq complexes correlate with the available structural and biochemical data. Our results have demonstrated that it is possible to determine single-stranded RNA-binding sites on the protein surface using a soaking procedure followed by structure determination of the nucleotide-protein complexes. In order to test our approach, RNA-binding proteins CspB from Bacillus subtilis and Rop from Escherichia coli were chosen. AMP and GMP affinities to the proteins have been also estimated by measuring of fluorescence changes during titration of the AMP-MANT and GMP-MANT solution by appropriate proteins.

 РНК-белковое узнавание рентгеноструктурный анализ регуляция транскрипции и трансляции флуоресцентно-меченые нуклеотиды RNA binding X-ray diffraction analysis transcription regulation translation regulation fluorescent analogs of nucleotides

Murina V.N., Lekontseva N.V., Nikulin A.D. Hfq binds ribonucleotides in three different RNA-binding sites. Acta Crystallogr. D Biol Crystallogr., 2013, vol. 69 (Pt8), pp.1504-13 Murina V.N., Lekontseva N.V., Nikulin A.D. Hfq binds ribonucleotides in three different RNA-binding sites. Acta Crystallogr. D Biol Crystallogr., 2013, vol. 69 (Pt8), pp.1504-13 Graumann P., Wendrich T.M., Weber M.H., Schroder K., Marahiel M.A. A family of cold shock proteins in Bacillus subtilis is essential for cellular growth and for efficient protein synthesis at optimal and low temperatures. Mol. Microbiol., 1997, vol. 25, pp. 741-756. Graumann P., Wendrich T.M., Weber M.H., Schroder K., Marahiel M.A. A family of cold shock proteins in Bacillus subtilis is essential for cellular growth and for efficient protein synthesis at optimal and low temperatures. Mol. Microbiol., 1997, vol. 25, pp. 741-756. Jiang W., Hou Y., Inouye M. CspA, the major cold-shock protein of Escherichia coli, is an RNA chaperone. J. Biol. Chem., 1997, vol. 272, pp. 196-202. Jiang W., Hou Y., Inouye M. CspA, the major cold-shock protein of Escherichia coli, is an RNA chaperone. J. Biol. Chem., 1997, vol. 272, pp. 196-202. Sachs R., Max K., Heinemann U., Balbach J. RNA single strands bind to a conserved surface of the major cold shock protein in crystals and solution. RNA, 2012, vol. 18, pp. 65-76. Sachs R., Max K., Heinemann U., Balbach J. RNA single strands bind to a conserved surface of the major cold shock protein in crystals and solution. RNA, 2012, vol. 18, pp. 65-76. Schindelin H., Herrler M., Willimsky G., Marahiel M.A., Heinemann U. Overproduction, crystallization, and preliminary X-ray diffraction studies of the major cold shock protein from Bacillus subtilis, CspB. Proteins: Struct. Funct. Genet., 1992, vol. 14, pp. 120-124. Schindelin H., Herrler M., Willimsky G., Marahiel M.A., Heinemann U. Overproduction, crystallization, and preliminary X-ray diffraction studies of the major cold shock protein from Bacillus subtilis, CspB. Proteins: Struct. Funct. Genet., 1992, vol. 14, pp. 120-124. Agrawal V., Radha Kishan K.V. OB fold: growing bigger with functional consistancy. Curr. Protein Peptide Sci., 2003, vol. 4, pp.195-206. Agrawal V., Radha Kishan K.V. OB fold: growing bigger with functional consistancy. Curr. Protein Peptide Sci., 2003, vol. 4, pp.195-206. Max K.E., Zeeb M., Bienert R., Balbach J., Heinemann U. T-rich DNA single strands bind to a preformed site on the bacterial cold shock protein Bs-CspB. J Mol Biol., 2006, vol. 360, pp. 702-714. Max K.E., Zeeb M., Bienert R., Balbach J., Heinemann U. T-rich DNA single strands bind to a preformed site on the bacterial cold shock protein Bs-CspB. J Mol Biol., 2006, vol. 360, pp. 702-714. Eguchi Y., Itoh T., Tomizawa J. Antisense RNA. Ann Rev Biochem., 1991, vol. 60, pp. 631-652. Eguchi Y., Itoh T., Tomizawa J. Antisense RNA. Ann Rev Biochem., 1991, vol. 60, pp. 631-652. Gregorian RS, Crothers DM. Determinants of RNA hairpin loop-loop complex stability. J Mol Biol., 1995, vol. 248, pp. 968-984. Gregorian RS, Crothers DM. Determinants of RNA hairpin loop-loop complex stability. J Mol Biol., 1995, vol. 248, pp. 968-984. Eguchi Y, Tomizawa J. Complexes formed by complementary RNA stem-loops--their formations, structures and interaction with ColE1 Rom protein. J Mol Biol., 1991, vol. 220, pp. 831-842. Eguchi Y, Tomizawa J. Complexes formed by complementary RNA stem-loops--their formations, structures and interaction with ColE1 Rom protein. J Mol Biol., 1991, vol. 220, pp. 831-842. Struble E.B., Ladner J.E., Brabazon D.M., Marino J.P. New crystal structures of ColE1 Rom and variants resulting from mutation of a surface exposed residue: Implications for RNA-recognition. Proteins, 2008, vol. 72, pp. 761-768. Struble E.B., Ladner J.E., Brabazon D.M., Marino J.P. New crystal structures of ColE1 Rom and variants resulting from mutation of a surface exposed residue: Implications for RNA-recognition. Proteins, 2008, vol. 72, pp. 761-768. Davies D., Segal D. Protein crystallization: microtechniques involving vapor diffusion. Methods Enzymol., 1971, vol. 22, pp. 266-269. Davies D., Segal D. Protein crystallization: microtechniques involving vapor diffusion. Methods Enzymol., 1971, vol. 22, pp. 266-269. Hulme E.C., Trevethick M.A. Ligand binding assays at equilibrium: Validation and interpretation, Br. J. Pharmacol., 2010, vol. 161, pp. 1219-1237. Hulme E.C., Trevethick M.A. Ligand binding assays at equilibrium: Validation and interpretation, Br. J. Pharmacol., 2010, vol. 161, pp. 1219-1237. Trott O., Olson A.J. Auto Dock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization and multithreading. Journal of computational chemistry, 2010, vol. 31, pp. 455-461. Trott O., Olson A.J. Auto Dock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization and multithreading. Journal of computational chemistry, 2010, vol. 31, pp. 455-461.