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Title: Crystallization and preliminary X-ray analysis of the mRNA-binding domain of elongation factor SelB from Escherichia coli in complex with RNA

Abstract

The mRNA-binding domain of E. coli selenocysteine-specific elongation factor SelB (residues 478–614; SelB-WH3/4) was overproduced in E. coli and its cognate mRNA ligand, 23 nucleotides of the SECIS RNA hairpin, was prepared by in vitro transcription. The purified SelB-WH3/4–SECIS RNA complex crystallized in space group C2 and diffracted to 2.3 Å. In bacteria, selenocysteine (the 21st amino acid) is incorporated into proteins via machinery that includes SelB, a specific translational elongation factor. SelB binds to an mRNA hairpin called the selenocysteine-insertion sequence (SECIS) and delivers selenocysteyl-tRNA{sup Sec} to the ribosomal A site. The minimum C-terminal fragment (residues 478–614) of Escherichia coli SelB (SelB-WH3/4) required for SECIS binding has been overexpressed and purified. This protein was crystallized in complex with 23 nucleotides of the SECIS hairpin at 294 K using the hanging-drop vapour-diffusion method. A data set was collected to 2.3 Å resolution from a single crystal at 100 K using ESRF beamline BM-30. The crystal belongs to space group C2, with unit-cell parameters a = 103.50, b = 56.51, c = 48.41 Å. The asymmetric unit contains one WH3/4-domain–RNA complex. The Matthews coefficient was calculated to be 3.37 Å{sup 3} Da{sup −1} and the solvent content was estimated to bemore » 67.4%.« less

Authors:
; ;  [1]
  1. Laboratoire de Chimie et Biologie Structurales, ICSN-CNRS, 1 Avenue de la Terrasse, 91190 Gif-sur-Yvette (France)
Publication Date:
OSTI Identifier:
22360335
Resource Type:
Journal Article
Resource Relation:
Journal Name: Acta Crystallographica. Section F; Journal Volume: 63; Journal Issue: Pt 5; Other Information: PMCID: PMC2335015; PMID: 17565186; PUBLISHER-ID: rp5001; OAI: oai:pubmedcentral.nih.gov:2335015; Copyright (c) International Union of Crystallography 2007; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United Kingdom
Language:
English
Subject:
75 CONDENSED MATTER PHYSICS, SUPERCONDUCTIVITY AND SUPERFLUIDITY; CRYSTALLIZATION; DIFFUSION; ELONGATION; ESCHERICHIA COLI; EUROPEAN SYNCHROTRON RADIATION FACILITY; IN VITRO; LIGANDS; MONOCRYSTALS; PROTEINS; RESOLUTION; SOLVENTS; SPACE GROUPS

Citation Formats

Soler, Nicolas, Fourmy, Dominique, and Yoshizawa, Satoko, E-mail: yoshizawa@icsn.cnrs-gif.fr. Crystallization and preliminary X-ray analysis of the mRNA-binding domain of elongation factor SelB from Escherichia coli in complex with RNA. United Kingdom: N. p., 2007. Web. doi:10.1107/S174430910701723X.
Soler, Nicolas, Fourmy, Dominique, & Yoshizawa, Satoko, E-mail: yoshizawa@icsn.cnrs-gif.fr. Crystallization and preliminary X-ray analysis of the mRNA-binding domain of elongation factor SelB from Escherichia coli in complex with RNA. United Kingdom. doi:10.1107/S174430910701723X.
Soler, Nicolas, Fourmy, Dominique, and Yoshizawa, Satoko, E-mail: yoshizawa@icsn.cnrs-gif.fr. Tue . "Crystallization and preliminary X-ray analysis of the mRNA-binding domain of elongation factor SelB from Escherichia coli in complex with RNA". United Kingdom. doi:10.1107/S174430910701723X.
@article{osti_22360335,
title = {Crystallization and preliminary X-ray analysis of the mRNA-binding domain of elongation factor SelB from Escherichia coli in complex with RNA},
author = {Soler, Nicolas and Fourmy, Dominique and Yoshizawa, Satoko, E-mail: yoshizawa@icsn.cnrs-gif.fr},
abstractNote = {The mRNA-binding domain of E. coli selenocysteine-specific elongation factor SelB (residues 478–614; SelB-WH3/4) was overproduced in E. coli and its cognate mRNA ligand, 23 nucleotides of the SECIS RNA hairpin, was prepared by in vitro transcription. The purified SelB-WH3/4–SECIS RNA complex crystallized in space group C2 and diffracted to 2.3 Å. In bacteria, selenocysteine (the 21st amino acid) is incorporated into proteins via machinery that includes SelB, a specific translational elongation factor. SelB binds to an mRNA hairpin called the selenocysteine-insertion sequence (SECIS) and delivers selenocysteyl-tRNA{sup Sec} to the ribosomal A site. The minimum C-terminal fragment (residues 478–614) of Escherichia coli SelB (SelB-WH3/4) required for SECIS binding has been overexpressed and purified. This protein was crystallized in complex with 23 nucleotides of the SECIS hairpin at 294 K using the hanging-drop vapour-diffusion method. A data set was collected to 2.3 Å resolution from a single crystal at 100 K using ESRF beamline BM-30. The crystal belongs to space group C2, with unit-cell parameters a = 103.50, b = 56.51, c = 48.41 Å. The asymmetric unit contains one WH3/4-domain–RNA complex. The Matthews coefficient was calculated to be 3.37 Å{sup 3} Da{sup −1} and the solvent content was estimated to be 67.4%.},
doi = {10.1107/S174430910701723X},
journal = {Acta Crystallographica. Section F},
number = Pt 5,
volume = 63,
place = {United Kingdom},
year = {Tue May 01 00:00:00 EDT 2007},
month = {Tue May 01 00:00:00 EDT 2007}
}