Structural characterization of nuclear poly(A)-protein particles in rat liver.

Poly(A)-protein particles were prepared from rat liver nuclear extract after digestion with pancreatic ribonuclease and ribonuclease T1 by sucrose gradient centrifugation. The particles were sedimented in a range of 9-23S with a peak at 16S. The particles isolated in this manner were 99-100% resistant to further pancreatic ribonuclease 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 corresponds 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 sulphate/polyacrylamide gel electrophoresis demonstrated two major polypeptide components with Mr of 63 000 and 90 000 and at least ten minor polypeptides in the 45 000-130 000-Mr range. In sodium dodecyl sulphate/polyacrylamide gels the 63 000-Mr polypeptide was the only one 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. On the basis of the results a model for the 'average' 16-S particle was constructed.