Altering the GTP binding site of the DNA/RNA-binding protein, Translin/TB-RBP, decreases RNA binding and may create a dominant negative phenotype.
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 approximately 50% and the poorly hydrolyzed GTP analog, GTPgammaS, 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-RBP(GTP) and TB-RBP(GTP) no longer binds to RNA, although DNA binding is not affected. Yeast two-hybrid assays reveal that like wild-type TB-RBP, TB-RBP(GTP) will interact with itself, with wild-type TB-RBP and with Translin associated factor X (Trax). Transfection of TB-RBP(GTP) into NIH 3T3 cells leads to a marked increase in cell death suggesting a dominant negative function for TB-RBP(GTP) in cells. These data suggest TB-RBP is an RNA-binding protein whose activity is allosterically controlled by nucleotide binding.
Chennathukuzhi
V M
VM
Center 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.
Kurihara
Y
Y
Bray
J D
JD
Yang
J
J
Hecht
N B
NB
D016475:3T3 Cells; D000494:Allosteric Regulation; D000495:Allosteric Site; D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism; D016923:Cell Death; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D005799:Genes, Dominant / Q000235:genetics*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006801:Humans; D008297:Male; D051379:Mice; D009154:Mutation / Q000235:genetics*; D009687:Nuclear Proteins / Q000378:metabolism; D010641:Phenotype; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012313:RNA / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013737:Testis / Q000378:metabolism; D020798:Two-Hybrid System Techniques
D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D016601:RNA-Binding Proteins; C095563:TSN protein, human; C498305:TSNAX protein, human; C498303:Tsn protein, mouse; C498306:Tsnax protein, mouse; D006153:Guanosine Diphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D012313:RNA; D006160:Guanosine Triphosphate; D004247:DNA