Local tertiary structure probing of ribonucleoprotein particles by nuclease fusion proteins.

Analyses of the conformational dynamics of the numerous cellular ribonucleoprotein particles (RNP) significantly contribute to the understanding of their modes of action. Here, we tested whether ribonuclease fusion proteins incorporated into RNPs can be used as molecular probes to characterize the l...

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Autores principales:	Uli Ohmayer, Jorge Perez-Fernandez, Thomas Hierlmeier, Gisela Pöll, Lydia Williams, Joachim Griesenbeck, Herbert Tschochner, Philipp Milkereit
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2012
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/d9dfe258a0374a7c832e6985175cc640
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spelling	oai:doaj.org-article:d9dfe258a0374a7c832e6985175cc6402021-11-18T07:09:55ZLocal tertiary structure probing of ribonucleoprotein particles by nuclease fusion proteins.1932-620310.1371/journal.pone.0042449https://doaj.org/article/d9dfe258a0374a7c832e6985175cc6402012-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/22876323/?tool=EBIhttps://doaj.org/toc/1932-6203Analyses of the conformational dynamics of the numerous cellular ribonucleoprotein particles (RNP) significantly contribute to the understanding of their modes of action. Here, we tested whether ribonuclease fusion proteins incorporated into RNPs can be used as molecular probes to characterize the local RNA environment of these proteins. Fusion proteins of micrococcal nuclease (MNase) with ribosomal proteins were expressed in S. cerevisae to produce in vivo recombinant ribosomes which have a ribonuclease tethered to specific sites. Activation of the MNase activity by addition of calcium led to specific rRNA cleavage events in proximity to the ribosomal binding sites of the fusion proteins. The dimensions of the RNP environment which could be probed by this approach varied with the size of the linker sequence between MNase and the fused protein. Advantages and disadvantages of the use of MNase fusion proteins for local tertiary structure probing of RNPs as well as alternative applications for this type of approach in RNP research are discussed.Uli OhmayerJorge Perez-FernandezThomas HierlmeierGisela PöllLydia WilliamsJoachim GriesenbeckHerbert TschochnerPhilipp MilkereitPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 7, Iss 8, p e42449 (2012)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Uli Ohmayer Jorge Perez-Fernandez Thomas Hierlmeier Gisela Pöll Lydia Williams Joachim Griesenbeck Herbert Tschochner Philipp Milkereit Local tertiary structure probing of ribonucleoprotein particles by nuclease fusion proteins.
description	Analyses of the conformational dynamics of the numerous cellular ribonucleoprotein particles (RNP) significantly contribute to the understanding of their modes of action. Here, we tested whether ribonuclease fusion proteins incorporated into RNPs can be used as molecular probes to characterize the local RNA environment of these proteins. Fusion proteins of micrococcal nuclease (MNase) with ribosomal proteins were expressed in S. cerevisae to produce in vivo recombinant ribosomes which have a ribonuclease tethered to specific sites. Activation of the MNase activity by addition of calcium led to specific rRNA cleavage events in proximity to the ribosomal binding sites of the fusion proteins. The dimensions of the RNP environment which could be probed by this approach varied with the size of the linker sequence between MNase and the fused protein. Advantages and disadvantages of the use of MNase fusion proteins for local tertiary structure probing of RNPs as well as alternative applications for this type of approach in RNP research are discussed.
format	article
author	Uli Ohmayer Jorge Perez-Fernandez Thomas Hierlmeier Gisela Pöll Lydia Williams Joachim Griesenbeck Herbert Tschochner Philipp Milkereit
author_facet	Uli Ohmayer Jorge Perez-Fernandez Thomas Hierlmeier Gisela Pöll Lydia Williams Joachim Griesenbeck Herbert Tschochner Philipp Milkereit
author_sort	Uli Ohmayer
title	Local tertiary structure probing of ribonucleoprotein particles by nuclease fusion proteins.
title_short	Local tertiary structure probing of ribonucleoprotein particles by nuclease fusion proteins.
title_full	Local tertiary structure probing of ribonucleoprotein particles by nuclease fusion proteins.
title_fullStr	Local tertiary structure probing of ribonucleoprotein particles by nuclease fusion proteins.
title_full_unstemmed	Local tertiary structure probing of ribonucleoprotein particles by nuclease fusion proteins.
title_sort	local tertiary structure probing of ribonucleoprotein particles by nuclease fusion proteins.
publisher	Public Library of Science (PLoS)
publishDate	2012
url	https://doaj.org/article/d9dfe258a0374a7c832e6985175cc640
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Local tertiary structure probing of ribonucleoprotein particles by nuclease fusion proteins.

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