Abstract
A direct, ribonuclease T1 protection assay was employed to study the binding of a delta agent genomic RNA transcript containing the conserved domain to the double-stranded RNA- (dsRNA-) dependent protein kinase of mammalian cells, PKR (also known as DAI, p1-elF2, or p68 kinase). In a control reaction, this assay identified a major portion of the same PKR binding site in VA RNA as deduced previously using a footprinting technique (Clarke PA, Mathews MB, 1995, RNA 1:1-20). Although delta agent RNA contains extensive secondary structure throughout the conserved region, we found a remarkable specificity in its PKR binding. The same region was protected by intact PKR and by a 184-amino acid fragment thereof containing the two RNA-binding motifs (dsRBMs) but lacking kinase activity. Two specific opposed, continuous segments of delta agent RNA (extending about 65-70 bases) were obtained reproducibly. Each is more than twice as long as those protected in VA RNA (about 25 bases), suggesting the involvement of PKR dimers in delta RNA binding. The PKR-protected region of delta agent RNA also contains a characteristic tertiary structural element that may be involved in binding specificity.
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