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Molecular and Cellular Biology, September 2007, p. 6350-6360, Vol. 27, No. 18
0270-7306/07/$08.00+0     doi:10.1128/MCB.00632-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

The L7Ae RNA Binding Motif Is a Multifunctional Domain Required for the Ribosome-Dependent Sec Incorporation Activity of Sec Insertion Sequence Binding Protein 2{triangledown}

Kelvin Caban, Scott A. Kinzy, and Paul R. Copeland*

Department of Molecular Genetics, Microbiology and Immunology, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway, New Jersey 08854

Received 11 April 2007/ Returned for modification 9 May 2007/ Accepted 10 July 2007

The decoding of specific UGA codons as selenocysteine is specified by the Sec insertion sequence (SECIS) element. Additionally, Sec-tRNA[Ser]Sec and the dedicated Sec-specific elongation factor eEFSec are required but not sufficient for nonsense suppression. SECIS binding protein 2 (SBP2) is also essential for Sec incorporation, but its precise role is unknown. In addition to binding the SECIS element, SBP2 binds stably and quantitatively to ribosomes. To determine the function of the SBP2-ribosome interaction, conserved amino acids throughout the SBP2 L7Ae RNA binding motif were mutated to alanine in clusters of five. Mutant proteins were analyzed for ribosome binding, SECIS element binding, and Sec incorporation activity, allowing us to identify two distinct but interdependent sites within the L7Ae motif: (i) a core L7Ae motif required for SECIS binding and ribosome binding and (ii) an auxiliary motif involved in physical and functional interactions with the ribosome. Structural modeling of SBP2 based on the 15.5-kDa protein-U4 snRNA complex strongly supports a two-site model for L7Ae domain function within SBP2. These results provide evidence that the SBP2-ribosome interaction is essential for Sec incorporation.

* Corresponding author. Mailing address: Department of Molecular Genetics, Microbiology and Immunology, UMDNJ-Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, NJ 08854. Phone: (732) 235-4670. Fax: (732) 235-5223. E-mail: paul.copeland{at}umdnj.edu

{triangledown} Published ahead of print on 16 July 2007.

Molecular and Cellular Biology, September 2007, p. 6350-6360, Vol. 27, No. 18
0270-7306/07/$08.00+0     doi:10.1128/MCB.00632-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Takeuchi, A., Schmitt, D., Chapple, C., Babaylova, E., Karpova, G., Guigo, R., Krol, A., Allmang, C. (2009). A short motif in Drosophila SECIS Binding Protein 2 provides differential binding affinity to SECIS RNA hairpins. Nucleic Acids Res 37: 2126-2141 [Abstract] [Full Text]  
  • Donovan, J., Caban, K., Ranaweera, R., Gonzalez-Flores, J. N., Copeland, P. R. (2008). A Novel Protein Domain Induces High Affinity Selenocysteine Insertion Sequence Binding and Elongation Factor Recruitment. J. Biol. Chem. 283: 35129-35139 [Abstract] [Full Text]  
  • Papp, L. V., Wang, J., Kennedy, D., Boucher, D., Zhang, Y., Gladyshev, V. N., Singh, R. N., Khanna, K. K. (2008). Functional characterization of alternatively spliced human SECISBP2 transcript variants. Nucleic Acids Res 36: 7192-7206 [Abstract] [Full Text]  


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