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Nucleic Acids Research, 2001, Vol. 29, No. 21 4433-4440
© 2001 Oxford University Press

Altering the GTP binding site of the DNA/RNA-binding protein, Translin/TB-RBP, decreases RNA binding and may create a dominant negative phenotype

Vargheese M. Chennathukuzhi1, Yasuyuki Kurihara1,2, Jeffrey D. Bray1, Juxiang Yang1 and Norman B. Hecht1,*

1Center for Research on Reproduction and Women’s Health and Department of Obstetrics and Gynecology, University of Pennsylvania School of Medicine, 1310 Biomedical Research Building II/III, 421 Curie Boulevard, Philadelphia, PA 19104-6142, USA and 2Yokohama National University, Graduate School of Environment and Information Sciences, Yokohama, Japan

The DNA/RNA-binding protein, Translin/Testis Brain RNA-binding protein (Translin/TB-RBP), contains a putative GTP binding site in its C-terminus which is highly conserved. To determine if guanine nucleotide binding to this site functionally alters nucleic acid binding, electrophoretic mobility shift assays were performed with RNA and DNA binding probes. GTP, but not GDP, reduces RNA binding by ~50% and the poorly hydrolyzed GTP analog, GTP{gamma}S, reduces binding by >90% in gel shift and immunoprecipitation assays. No similar reduction of DNA binding is seen. When the putative GTP binding site of TB-RBP, amino acid sequence VTAGD, is altered to VTNSD by site directed mutagenesis, GTP will no longer bind to TB-RBPGTP and TB-RBPGTP no longer binds to RNA, although DNA binding is not affected. Yeast two-hybrid assays reveal that like wild-type TB-RBP, TB-RBPGTP will interact with itself, with wild-type TB-RBP and with Translin associated factor X (Trax). Transfection of TB-RBPGTP into NIH 3T3 cells leads to a marked increase in cell death suggesting a dominant negative function for TB-RBPGTP in cells. These data suggest TB-RBP is an RNA-binding protein whose activity is allosterically controlled by nucleotide binding.

* To whom correspondence should be addressed. Tel: +1 215 898 0144; Fax: +1 215 573 5408; Email: nhecht{at}mail.med.upenn.edu


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