Spatial Distribution and Ribosome-Binding Dynamics of EF-P in Live <italic toggle="yes">Escherichia coli</italic>

ABSTRACT In vitro assays find that ribosomes form peptide bonds to proline (Pro) residues more slowly than to other residues. Ribosome profiling shows that stalling at Pro-Pro-X triplets is especially severe but is largely alleviated in Escherichia coli by the action of elongation factor EF-P. EF-P...

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Autores principales:	Sonisilpa Mohapatra, Heejun Choi, Xueliang Ge, Suparna Sanyal, James C. Weisshaar
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2017
Materias:	EF-P binding dynamics live E. coli superresolution fluorescence Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/10a425c6ed444e09a6ecc246fef46bd4
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spelling	oai:doaj.org-article:10a425c6ed444e09a6ecc246fef46bd42021-11-15T15:51:29ZSpatial Distribution and Ribosome-Binding Dynamics of EF-P in Live <italic toggle="yes">Escherichia coli</italic>10.1128/mBio.00300-172150-7511https://doaj.org/article/10a425c6ed444e09a6ecc246fef46bd42017-07-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.00300-17https://doaj.org/toc/2150-7511ABSTRACT In vitro assays find that ribosomes form peptide bonds to proline (Pro) residues more slowly than to other residues. Ribosome profiling shows that stalling at Pro-Pro-X triplets is especially severe but is largely alleviated in Escherichia coli by the action of elongation factor EF-P. EF-P and its eukaryotic/archaeal homolog IF5A enhance the peptidyl transfer step of elongation. Here, a superresolution fluorescence localization and tracking study of EF-P–mEos2 in live E. coli provides the first in vivo information about the spatial distribution and on-off binding kinetics of EF-P. Fast imaging at 2 ms/frame helps to distinguish ribosome-bound (slowly diffusing) EF-P from free (rapidly diffusing) EF-P. Wild-type EF-P exhibits a three-peaked axial spatial distribution similar to that of ribosomes, indicating substantial binding. The mutant EF-PK34A exhibits a homogeneous distribution, indicating little or no binding. Some 30% of EF-P copies are bound to ribosomes at a given time. Two-state modeling and copy number estimates indicate that EF-P binds to 70S ribosomes during 25 to 100% of translation cycles. The timescale of the typical diffusive search by free EF-P for a ribosome-binding site is τfree ≈ 16 ms. The typical residence time of an EF-P on the ribosome is very short, τbound ≈ 7 ms. Evidently, EF-P binds to ribosomes during many or most elongation cycles, much more often than the frequency of Pro-Pro motifs. Emptying of the E site during part of the cycle is consistent with recent in vitro experiments indicating dissociation of the deacylated tRNA upon translocation. IMPORTANCE Ribosomes translate the codon sequence within mRNA into the corresponding sequence of amino acids within the nascent polypeptide chain, which in turn ultimately folds into functional protein. At each codon, bacterial ribosomes are assisted by two well-known elongation factors: EF-Tu, which aids binding of the correct aminoacyl-tRNA to the ribosome, and EF-G, which promotes tRNA translocation after formation of the new peptide bond. A third factor, EF-P, has been shown to alleviate ribosomal pausing at rare Pro-Pro motifs, which are translated very slowly without EF-P. Here, we use superresolution fluorescence imaging to study the spatial distribution and ribosome-binding dynamics of EF-P in live E. coli cells. We were surprised to learn that EF-P binds to and unbinds from translating ribosomes during at least 25% of all elongation events; it may bind during every elongation cycle.Sonisilpa MohapatraHeejun ChoiXueliang GeSuparna SanyalJames C. WeisshaarAmerican Society for MicrobiologyarticleEF-Pbinding dynamicslive E. colisuperresolution fluorescenceMicrobiologyQR1-502ENmBio, Vol 8, Iss 3 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	EF-P binding dynamics live E. coli superresolution fluorescence Microbiology QR1-502
spellingShingle	EF-P binding dynamics live E. coli superresolution fluorescence Microbiology QR1-502 Sonisilpa Mohapatra Heejun Choi Xueliang Ge Suparna Sanyal James C. Weisshaar Spatial Distribution and Ribosome-Binding Dynamics of EF-P in Live <italic toggle="yes">Escherichia coli</italic>
description	ABSTRACT In vitro assays find that ribosomes form peptide bonds to proline (Pro) residues more slowly than to other residues. Ribosome profiling shows that stalling at Pro-Pro-X triplets is especially severe but is largely alleviated in Escherichia coli by the action of elongation factor EF-P. EF-P and its eukaryotic/archaeal homolog IF5A enhance the peptidyl transfer step of elongation. Here, a superresolution fluorescence localization and tracking study of EF-P–mEos2 in live E. coli provides the first in vivo information about the spatial distribution and on-off binding kinetics of EF-P. Fast imaging at 2 ms/frame helps to distinguish ribosome-bound (slowly diffusing) EF-P from free (rapidly diffusing) EF-P. Wild-type EF-P exhibits a three-peaked axial spatial distribution similar to that of ribosomes, indicating substantial binding. The mutant EF-PK34A exhibits a homogeneous distribution, indicating little or no binding. Some 30% of EF-P copies are bound to ribosomes at a given time. Two-state modeling and copy number estimates indicate that EF-P binds to 70S ribosomes during 25 to 100% of translation cycles. The timescale of the typical diffusive search by free EF-P for a ribosome-binding site is τfree ≈ 16 ms. The typical residence time of an EF-P on the ribosome is very short, τbound ≈ 7 ms. Evidently, EF-P binds to ribosomes during many or most elongation cycles, much more often than the frequency of Pro-Pro motifs. Emptying of the E site during part of the cycle is consistent with recent in vitro experiments indicating dissociation of the deacylated tRNA upon translocation. IMPORTANCE Ribosomes translate the codon sequence within mRNA into the corresponding sequence of amino acids within the nascent polypeptide chain, which in turn ultimately folds into functional protein. At each codon, bacterial ribosomes are assisted by two well-known elongation factors: EF-Tu, which aids binding of the correct aminoacyl-tRNA to the ribosome, and EF-G, which promotes tRNA translocation after formation of the new peptide bond. A third factor, EF-P, has been shown to alleviate ribosomal pausing at rare Pro-Pro motifs, which are translated very slowly without EF-P. Here, we use superresolution fluorescence imaging to study the spatial distribution and ribosome-binding dynamics of EF-P in live E. coli cells. We were surprised to learn that EF-P binds to and unbinds from translating ribosomes during at least 25% of all elongation events; it may bind during every elongation cycle.
format	article
author	Sonisilpa Mohapatra Heejun Choi Xueliang Ge Suparna Sanyal James C. Weisshaar
author_facet	Sonisilpa Mohapatra Heejun Choi Xueliang Ge Suparna Sanyal James C. Weisshaar
author_sort	Sonisilpa Mohapatra
title	Spatial Distribution and Ribosome-Binding Dynamics of EF-P in Live <italic toggle="yes">Escherichia coli</italic>
title_short	Spatial Distribution and Ribosome-Binding Dynamics of EF-P in Live <italic toggle="yes">Escherichia coli</italic>
title_full	Spatial Distribution and Ribosome-Binding Dynamics of EF-P in Live <italic toggle="yes">Escherichia coli</italic>
title_fullStr	Spatial Distribution and Ribosome-Binding Dynamics of EF-P in Live <italic toggle="yes">Escherichia coli</italic>
title_full_unstemmed	Spatial Distribution and Ribosome-Binding Dynamics of EF-P in Live <italic toggle="yes">Escherichia coli</italic>
title_sort	spatial distribution and ribosome-binding dynamics of ef-p in live <italic toggle="yes">escherichia coli</italic>
publisher	American Society for Microbiology
publishDate	2017
url	https://doaj.org/article/10a425c6ed444e09a6ecc246fef46bd4
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Spatial Distribution and Ribosome-Binding Dynamics of EF-P in Live <italic toggle="yes">Escherichia coli</italic>

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