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Lookup NU author(s): Dr Heather Lamb,
Dr Nick Watkins,
Professor Alastair Hawkins
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The S. typhimurium genome encodes proteins, designated EngA and YhbZ, which have a high sequence identity with the GTPases EngA/Der and ObgE/CgtA E of Escherichia coli. The wild-type activity of the E. coli proteins is essential for normal ribosome maturation and cell viability. In order to characterize the potential involvement of the Salmonella typhimurium EngA and YhbZ proteins in ribosome biology, we used high stringency affinity chromatography experiments to identify strongly binding ribosomal partner proteins. A combination of biochemical and microcalorimetric analysis was then used to characterize these protein:protein interactions and quantify nucleotide binding affinities. These experiments show that YhbZ specifically interacts with the pseudouridine synthase RluD (KD = 2 μM and 1:1 stoichiometry), and we show for the first time that EngA can interact with the ribosomal structural protein S7. Thermodynamic analysis shows both EngA and YhbZ bind GDP with a higher affinity than GTP (20-fold difference for EngA and 3.8-fold for YhbZ), and that the two nucleotide binding sites in EngA show a 5.3-fold difference in affinity for GDP. We report a fluorescence assay for nucleotide binding to EngA and YhbZ, which is suitable for identifying inhibitors specific for this ligand-binding site, which would potentially inhibit their biological functions. The interactions of YhbZ with ribosome structural proteins that we identify may demonstrate a previously unreported additional function for this class of GTPase: that of ensuring delivery of rRNA modifying enzymes to the appropriate region of the ribosome. Published by Cold Spring Harbor Laboratory Press. Copyright © 2007 The Protein Society.
Author(s): Lamb HK, Thompson P, Elliott C, Charles IG, Richards J, Lockyer M, Watkins N, Nichols C, Stammers DK, Bagshaw CR, Cooper A, Hawkins AR
Publication type: Article
Publication status: Published
Journal: Protein Science
ISSN (print): 0961-8368
ISSN (electronic): 1469-896X
Publisher: Wiley-Blackwell Publishing, Inc.
PubMed id: 17905831
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