Interactions Of The A1 Heterogeneous Nuclear Ribonucleoprotein And Its Proteolytic Derivative, Up1, With Rna And Dna - Evidence For Multiple Rna-Binding Domains And Salt-Dependent Binding Mode Transitions

The 319-residue A1 heterogeneous nuclear ribonucleoprotein is the best studied of the group of major or core mammalian hnRNP proteins that bind pre-mRNA immediately following transcription. Circular dichroism studies suggest that binding of A1 and its proteolytic fragment, UP1 (residues 1-195), to nucleic acids results in an unstacking of the bases of poly(A). On the basis of poly[d(A-T)] and poly[r(A-U)] melting studies, both A1 and UP1 are helix-destabilizing proteins. Titrations of A1 and UP1 with poly(A), poly(U), and poly[d(T)] suggest that these two proteins do not bind with significant base specificity. A previous study indicated that A1, which contains a glycine-rich COOH terminus (residues 196-319) not present in UP1, binds cooperatively to polynucleotides while UP1 does not [Cobianchi et al. (1988) J. Biol. Chem. 263, 1063-1071]. Here we confirm this latter finding and demonstrate that the cooperativity parameter for A1 binding, which has a value of about 35 for binding to both single-stranded RNA and DNA, is insensitive to the NaCl concentration at least up to 0.4 M. In contrast to the cooperativity parameter, the occluded site size for A1 binding to RNA is salt dependent and increases from about 14 to 28 upon increasing the NaCl concentration from 25 to 250 mM. This variation in site size is best explained by assuming that A1 can interact with nucleic acids via at least two different binding modes. Both A1 and UP1 have higher affinity for single-stranded as opposed to double-stranded nucleic acids and bind preferentially to single-stranded RNA as compared to DNA. Comparative studies on the binding of A1 versus UP1 to poly[r(epsilon-A)] demonstrate that in addition to cooperative protein/protein interactions, the glycine-rich COOH-terminal domain of A1 is also directly involved in protein/nucleic acid interactions. These conclusions are directly confirmed by studies on a 48-residue synthetic peptide corresponding to residues 260-307 in A1. In 10 mM NaCl, this peptide has an intrinsic affinity of 1.5 X 10(6) M-1 for poly[r(epsilon-A)] and a cooperativity parameter of about 30. The similar cooperativity parameters for A1 and this synthetic peptide analogue suggests that most of the structural determinants necessary for A1/A1 cooperative protein interactions are within residues 260-307 in A1. Together these studies demonstrate that the UP1 and glycine-rich COOH-terminal domains of A1 make an approximately equal and independent contribution to the overall free energy of A1 binding to single-stranded RNA.