title: Purification and nucleic acid binding properties of a fragment of type C1/C2 heterogeneous nuclear ribonucleoprotein from thymic nuclear extracts.
abstract: A single-strand nucleic acid binding protein (C/F) that has an apparent molecular weight of 12,000 on SDS-polyacrylamide gel electrophoresis and that was originally thought to be the 12-kDa alpha-subunit of the AB form of terminal deoxynucleotidyl transferase (TdT) from calf thymus has been purified and identified as a fragment of the type C1/C2 hnRNP proteins. On the basis of NH2-terminal sequencing and mass spectrometric analysis, C/F contains approximately 94 residues and spans from residue 9 to approximately residue 102 in the type C1/C2 hnRNP proteins. C/F is presumably produced in vitro via limited proteolysis of the type C1/C2 hnRNP proteins following cell disruption. Since C/F corresponds almost exactly to the approximately 90-residue conserved ribonucleoprotein binding domain (RBD) that is shared by many eukaryotic RNA binding proteins, it provided an opportunity to better characterize the domain structure of the type C1/C2 hnRNP proteins and to compare the nucleic acid binding properties of the type C1/C2 and A1 [see Shamoo et al. (1994) Biochemistry, preceding paper in this issue] RNA binding domains. Like the type A1 RBD, the type C1/C2 RBD has an apparent occluded site size of 6-7 nucleotides. The type C1/C2 RBD binds non-cooperatively to homopolynucleotides and has preferential affinity for RNA and for single as opposed to double-stranded nucleic acids. The type C1/C2 RBD has about a 100-fold higher affinity than the type A1 RBD does for RNA and some of this increased affinity results from additional ionic interactions. The latter account for approximately 50% of the free energy of binding of the type C1/C2 RBD. While the type C1/C2 hnRNP proteins exist in vivo as a very tight tetramer with the structure (C1)3C2 [Barnett et al. (1989) Mol. Cell. Biol. 9, 492-498], the isolated type C1/C2 RBD is a monomer. Hence, the determinants for tetramerization appear to lie outside the type C1/C2 RBD. Phenylalanine 19 was identified as the only point of photochemical cross-linking of the type C1/C2 RBD to [d(T)]8. This residue corresponds to the major site of cross-linking of the A1 RBD to [d(T)]8 [Merrill, B. M., Stone, K. L., Cobianchi, F., Wilson, S. H., & Williams, K. R. (1988) J. Biol. Chem. 263, 3307-3313].(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Department of Biochemistry and Molecular Biology, UMDNJ-New Jersey Medical School, Newark 07103.
authors.forename: S B
authors.initials: SB
authors.lastname: Amrute
authors.forename: Z
authors.initials: Z
authors.lastname: Abdul-Manan
authors.forename: V
authors.initials: V
authors.lastname: Pandey
authors.forename: K R
authors.initials: KR
authors.lastname: Williams
authors.forename: M J
authors.initials: MJ
authors.lastname: Modak
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002467:Cell Nucleus / Q000737:chemistry; D002845:Chromatography; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents; D003488:Cyanogen Bromide; D004277:DNA, Single-Stranded / Q000378:metabolism*; D000076245:Heterogeneous Nuclear Ribonucleoprotein A1; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B; D034482:Heterogeneous-Nuclear Ribonucleoprotein Group C; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D012313:RNA / Q000378:metabolism*; D012261:Ribonucleoproteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013950:Thymus Gland / Q000737:chemistry* / Q000648:ultrastructure; D014466:Ultraviolet Rays
chemical_list: C439788:C1 HNRNP; D003432:Cross-Linking Reagents; D004277:DNA, Single-Stranded; D000076245:Heterogeneous Nuclear Ribonucleoprotein A1; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B; D034482:Heterogeneous-Nuclear Ribonucleoprotein Group C; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments; D012261:Ribonucleoproteins; C027903:oligo (dT); D012313:RNA; D003488:Cyanogen Bromide