Structural Characterization of Nuclear Poly(A)-Protein Particles in Rat Liver

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Abstract
Poly(A)-protein particles were prepared from rat liver nuclear extract after digestion with pancreatic RNase [EC 3.1.27.5] and RNase T1 [EC 3.1.27.3] by sucrose gradient centrifugation. The particles were sedimented in a range of 9-23 S with a peak at 16 S. The particles isolated in this manner were 99-100% resistant to further pancreatic RNase treatment and contained more than 90% adenylic acid. In CsCl density gradient the nuclear poly(A)-protein particles banded in a narrow density range of 1.28-1.32 g/cm3 with a peak at 1.30 g/cm3, which corrresponds to about 90% of protein in the particles. The average length of the poly(A) molecules prepared from the 16-S particles was about 140 nucleotides. Urea/sodium dodecyl sulfate [SDS]/polyacrylamide gel electrophoresis demonstrated 2 major polypeptide components with MW of 63,000 and 90,000 and at least 10 minor polypeptides in the 45,000-130,000-MW range. In SDS/polyacrylamide gels the 63,000-MW polypeptide was the only 1 major component. Amino acid analysis of the polypeptides bound to nuclear poly(A) revealed that the polypeptides contained a relatively large amount of aspartic acid + asparagine and glutamic acid + glutamine (24%). Treatment of glutaraldehyde-fixed particles with micrococcal nuclease showed that more than 90% of the poly(A) was accessible to the enzyme, thus almost the entire poly(A) should be located on the surface of the particles. A model for the average 16-S particle was constructed.