Characterization of the DNA-Binding Domain of the Avian Y-Box Protein, chkYB-2, and Mutational Analysis of Its Single-Strand Binding Motif in the Rous Sarcoma Virus Enhancer

chkYB-2 is a sequence-specific, single-stranded DNA binding chicken Y-box protein that promotes Rous sarcoma virus long terminal repeat (RSV LTR)-driven transcription in avian fibroblasts. The DNA-binding domain of chkYB-2 has been mapped by characterizing the DNA binding properties of purified recombinant chkYB-2 mutant polypeptides. The data indicate that the invariant cold shock domain (CSD) is necessary but not sufficient for association with DNA and suggest that another conserved region, adjacent to the carboxyl boundary of the CSD, plays a role in high-affinity DNA binding. chkYB-2 binds to a tandem repeat of the 5*-GTACCACC-3* motif on the RSV LTR. Mutational analysis of this recognition sequence revealed the requirement of an essentially unaltered template for both high-affinity binding by chkYB-2 as well as maximal transcriptional activity of the RSV LTR in vivo. The single-stranded DNA binding activity of chkYB-2 is augmented by Mg 21 . The possible significance of this finding for transactivation by a single-strand DNA binding protein is discussed. The chicken Y-box protein, chkYB-2, is a sequence-specific single-stranded DNA binding protein that binds the octanucleotide motif 59-GTACCACC-39 present on the noncoding strand of the Rous sarcoma virus (RSV) long terminal repeat (LTR) (7). chkYB-2 is expressed abundantly in avian fibroblasts and muscle tissue, the mesenchymal-lineage host cells most permissive to infection and tumor formation by RSV. This property combined with its ability to function as a potent activator of RSV LTR-driven transcription in avian fibroblasts suggests an important role for this protein in the virus life cycle (40). We reported earlier on the cloning and characterization