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Nucleic Acids Research Advance Access originally published online on September 4, 2009
Nucleic Acids Research 2009 37(19):6503-6514; doi:10.1093/nar/gkp711
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Nucleic Acids Research, 2009, Vol. 37, No. 19 6503-6514
© The Author(s) 2009. Published by Oxford University Press.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.5/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Nucleic Acid Enzymes

A dominant negative mutant of the E. coli RNA helicase DbpA blocks assembly of the 50S ribosomal subunit

Lisa M. Sharpe Elles1, Michael T. Sykes2, James R. Williamson2 and Olke C. Uhlenbeck1,*

1Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, IL, 60208, USA and 2Department of Molecular Biology and Department of Chemistry, The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037, USA

*To whom correspondence should be addressed. Tel: +1 847 491 5139; Fax: +1 847 491 5444; Email: o-uhlenbeck{at}northwestern.edu

Received April 17, 2009. Revised August 10, 2009. Accepted August 11, 2009.

Escherichia coli DbpA is an ATP-dependent RNA helicase with specificity for hairpin 92 of 23S ribosomal RNA, an important part of the peptidyl transferase center. The R331A active site mutant of DbpA confers a dominant slow growth and cold sensitive phenotype when overexpressed in E. coli containing endogenous DbpA. Ribosome profiles from cells overexpressing DbpA R331A display increased levels of 50S and 30S subunits and decreased levels 70S ribosomes. Profiles run at low Mg2+ exhibit fewer 50S subunits and accumulate a 45S particle that contains incompletely processed and undermodified 23S rRNA in addition to reduced levels of several ribosomal proteins that bind late in the assembly pathway. Unlike mature 50S subunits, these 45S particles can stimulate the ATPase activity of DbpA, indicating that hairpin 92 has not yet been sequestered within the 50S subunit. Overexpression of the inactive DbpA R331A mutant appears to block assembly at a late stage when the peptidyl transferase center is formed, indicating a possible role for DbpA promoting this conformational change.

Present address: Lisa M. Sharpe Elles, Department of Chemistry, Washburn University, Topeka, KS 66621, USA.


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