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Molecular and Cellular Biology, February 2006, p. 1195-1208, Vol. 26, No. 4
0270-7306/06/$08.00+0     doi:10.1128/MCB.26.4.1195-1208.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Comprehensive Mutational Analysis of Yeast DEXD/H Box RNA Helicases Involved in Large Ribosomal Subunit Biogenesis

Kara A. Bernstein,2 Sander Granneman,1 Alicia V. Lee,1 Swarnameenakshi Manickam,1 and Susan J. Baserga1,2,3*

Departments of Molecular Biophysics and Biochemistry,1 Genetics,2 Therapeutic Radiology, Yale University School of Medicine, New Haven, Connecticut 065203

Received 25 July 2005/ Returned for modification 19 August 2005/ Accepted 15 November 2005

DEXD/H box putative RNA helicases are required for pre-rRNA processing in Saccharomyces cerevisiae, although their exact roles and substrates are unknown. To characterize the significance of the conserved motifs for helicase function, a series of five mutations were created in each of the eight essential RNA helicases (Has1, Dbp6, Dbp10, Mak5, Mtr4, Drs1, Spb4, and Dbp9) involved in 60S ribosomal subunit biogenesis. Each mutant helicase was screened for the ability to confer dominant negative growth defects and for functional complementation. Different mutations showed different degrees of growth inhibition among the helicases, suggesting that the conserved regions do not function identically in vivo. Mutations in motif I and motif II (the DEXD/H box) often conferred dominant negative growth defects, indicating that these mutations do not interfere with substrate binding. In addition, mutations in the putative unwinding domains (motif III) demonstrated that conserved amino acids are often not essential for function. Northern analysis of steady-state RNA from strains expressing mutant helicases showed that the dominant negative mutations also altered pre-rRNA processing. Coimmunoprecipitation experiments indicated that some RNA helicases associated with each other. In addition, we found that yeasts disrupted in expression of the two nonessential RNA helicases, Dbp3 and Dbp7, grew worse than when either one alone was disrupted.

* Corresponding author. Mailing address: Molecular Biophysics & Biochemistry Department, Yale University School of Medicine, 333 Cedar St., SHM C-114, New Haven, CT 06520-8024. Phone: (203) 785-4618. Fax: (203) 785-6404. E-mail: susan.baserga{at}yale.edu.

Molecular and Cellular Biology, February 2006, p. 1195-1208, Vol. 26, No. 4
0022-538X/06/$08.00+0     doi:10.1128/MCB.26.4.1195-1208.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Bernstein, J., Patterson, D. N., Wilson, G. M., Toth, E. A. (2008). Characterization of the Essential Activities of Saccharomyces cerevisiae Mtr4p, a 3'->5' Helicase Partner of the Nuclear Exosome. J. Biol. Chem. 283: 4930-4942 [Abstract] [Full Text]  
  • Martin-Marcos, P., Hinnebusch, A. G., Tamame, M. (2007). Ribosomal Protein L33 Is Required for Ribosome Biogenesis, Subunit Joining, and Repression of GCN4 Translation. Mol. Cell. Biol. 27: 5968-5985 [Abstract] [Full Text]  
  • Linder, P. (2006). Dead-box proteins: a family affair--active and passive players in RNP-remodeling. Nucleic Acids Res 34: 4168-4180 [Abstract] [Full Text]  
  • Granneman, S., Lin, C., Champion, E. A., Nandineni, M. R., Zorca, C., Baserga, S. J. (2006). The nucleolar protein Esf2 interacts directly with the DExD/H box RNA helicase, Dbp8, to stimulate ATP hydrolysis. Nucleic Acids Res 34: 3189-3199 [Abstract] [Full Text]  
  • Granneman, S., Bernstein, K. A., Bleichert, F., Baserga, S. J. (2006). Comprehensive Mutational Analysis of Yeast DEXD/H Box RNA Helicases Required for Small Ribosomal Subunit Synthesis. Mol. Cell. Biol. 26: 1183-1194 [Abstract] [Full Text]  


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