The long term goal of this project is to obtain a mechanistic understanding of a large, ubiquitous class of proteins, termed DEXD/H proteins, which participate as essential factors in many cellular processes involving RNA. DExD/H proteins are believed to act as RNA helicases to catalyze conformational changes in large RNAs, however, other functions have been proposed. The intent is to perform biochemical and biophysical experiments on purified proteins that will complement extensive efforts by many other groups applying molecular genetic and molecular biological methods to the same proteins in their more complex physiological setting. This project initially chose E. coli DbpA as a model for detailed study, because of its exceptional experimental tractability. Not only is it biochemically well-behaved, but, in contrast with all other DExD/H proteins, it shows very tight binding and high specificity for its target RNA, which simplifies structural and biochemical experiments. We have established that DbpA interacts with RNA in a unique manner, shown that it has helicase activity and understood how the high affinity and specificity is achieved. Current aims include (1) mechanistic experiments to understand how DbpA acts as a helicase and whether it is designed to only open a few base pairs. (2) biochemical experiments defining how DbpA interacts with 23S rRNA and (3) molecular microbiological experiments designed to determine the step in the bacterial ribosome assembly pathway where DbpA acts. Finally, high throughput RNA binding, ATPase and RNA helicase assays in microtiter plates will be developed, in order to assay many other DExD/H proteins, including the 18 family members involved in yeast ribosome assembly.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM060268-08
Application #
7263029
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Program Officer
Preusch, Peter C
Project Start
2000-06-01
Project End
2009-07-31
Budget Start
2007-08-01
Budget End
2009-07-31
Support Year
8
Fiscal Year
2007
Total Cost
$241,009
Indirect Cost
Name
Northwestern University at Chicago
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
160079455
City
Evanston
State
IL
Country
United States
Zip Code
60201
Garcia, Ivelitza; Albring, Michael J; Uhlenbeck, Olke C (2012) Duplex destabilization by four ribosomal DEAD-box proteins. Biochemistry 51:10109-18
Sharpe Elles, Lisa M; Sykes, Michael T; Williamson, James R et al. (2009) A dominant negative mutant of the E. coli RNA helicase DbpA blocks assembly of the 50S ribosomal subunit. Nucleic Acids Res 37:6503-14
Garcia, Ivelitza; Uhlenbeck, Olke C (2008) Differential RNA-dependent ATPase activities of four rRNA processing yeast DEAD-box proteins. Biochemistry 47:12562-73
Elles, Lisa M Sharpe; Uhlenbeck, Olke C (2008) Mutation of the arginine finger in the active site of Escherichia coli DbpA abolishes ATPase and helicase activity and confers a dominant slow growth phenotype. Nucleic Acids Res 36:41-50
Wang, Shuying; Hu, Yaoxiong; Overgaard, Michael T et al. (2006) The domain of the Bacillus subtilis DEAD-box helicase YxiN that is responsible for specific binding of 23S rRNA has an RNA recognition motif fold. RNA 12:959-67
Polach, Kevin J; Uhlenbeck, Olke C (2002) Cooperative binding of ATP and RNA substrates to the DEAD/H protein DbpA. Biochemistry 41:3693-702
Tsu, C A; Kossen, K; Uhlenbeck, O C (2001) The Escherichia coli DEAD protein DbpA recognizes a small RNA hairpin in 23S rRNA. RNA 7:702-9
Diges, C M; Uhlenbeck, O C (2001) Escherichia coli DbpA is an RNA helicase that requires hairpin 92 of 23S rRNA. EMBO J 20:5503-12