Enzima RNA helicase modificada em Escherichia coli

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. Uhlenbeck^{1,* 1}Department 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@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.

Tradução by bolha!!!!
A Escherichia coli DbpA é uma ATP dependente da enzima RNA helicase com especificidade para ... RNA ribossomal, uma importante parte central da enzima peptidil transferase. O R331A ativa um local de mutação da DbpA confere uma dominancia e um crescimento lento e  um fenótipo de sensibilidade ai frio, quando superexpressa em E. coli contaminada.

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