Studies on a nucleoprotein prepared from rat liver polysomes by digestion with T1 ribonuclease.
1. Treatment of rat liver polysomes in a buffer containing 2.5mm-magnesium chloride with T(1) ribonuclease at a concentration of 330units/ml. of reaction medium at 37 degrees for 2hr. leads to the production of an insoluble nucleoprotein. 2. On the bases of analysis for protein and RNA and of u.v.-absorption spectra the nucleoprotein appears to have lost approx. 60% of the structural RNA originally present in the ribosome. Degradation of (3)H-labelled polysomes (structural RNA labelled with orotic acid) with T(1) ribonuclease leads to nucleoprotein preparations retaining approx. 30% of the radioactivity originally present in the polysomes. By means of sucrose-density-gradient centrifugation it is shown that the nucleoprotein preparations are free of single 73s ribosomes and ribosomal subunits. No evidence for the presence of 28s and 18s structural RNA was obtained on examination of extracted nucleoprotein-particle RNA by means of sucrose-density-gradient centrifugation. 3. Digestion of washed polysomes carrying (14)C-labelled nascent peptide chains with T(1) ribonuclease gives a nucleoprotein particle that retains approx. 70% of the original labelled chains. Treatment of labelled nucleoprotein particles with 1mm-puromycin in the absence of transfer factors releases 20% of the labelled chains. Addition of GTP (0.48mumole) increases this release to 37%. 4. Treatment of nucleoprotein particles carrying (14)C-labelled peptide chains with either EDTA (50mm) or ammonium chloride (0.5m) brings about a small release of labelled material (approx. 15%). 5. Disruption of nucleoprotein particles carrying (14)C-labelled peptide chains with either sodium dodecyl sulphate or 2m-lithium chloride, followed by addition of transfer RNA as marker and chromatography on Sephadex G-200, show in both cases that considerable amounts of labelled peptide material move well ahead of the added transfer RNA marker. Further, if nucleoprotein particles carrying labelled peptide chains are treated with 0.3m-potassium hydroxide at 20 degrees for 24 hr., neutralized to pH7.6, and then chromatographed on Sephadex G-200, the labelled peptide material moves much closer to the added transfer RNA marker. These results suggest that a proportion of the nascent (14)C-labelled peptides on the nucleoprotein are attached to transfer RNA or large fragments of transfer RNA. 6. [(3)H]Polyuridylic acid binds to nucleoprotein particles in 1mm-magnesium chloride. The rate of binding is rapid when measured at 20 degrees .
D000643:Ammonium Chloride; D000818:Animals; D002247:Carbon Isotopes; D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel; D003902:Detergents; D004492:Edetic Acid; D006150:Guanine Nucleotides; D008025:Ligases; D008099:Liver / Q000166:cytology; D009698:Nucleoproteins; D009963:Orotic Acid; D010455:Peptides; D011119:Polynucleotides; D011485:Protein Binding; D011691:Puromycin; D012313:RNA / Q000032:analysis; D012343:RNA, Transfer; D051381:Rats; D012260:Ribonucleases; D012270:Ribosomes / Q000032:analysis*; D013057:Spectrum Analysis; D014316:Tritium; D014466:Ultraviolet Rays; D014500:Uracil Nucleotides
D002247:Carbon Isotopes; D003902:Detergents; D006150:Guanine Nucleotides; D009698:Nucleoproteins; D010455:Peptides; D011119:Polynucleotides; D014500:Uracil Nucleotides; D000643:Ammonium Chloride; D014316:Tritium; D011691:Puromycin; D009963:Orotic Acid; D012313:RNA; D012343:RNA, Transfer; D004492:Edetic Acid; D012260:Ribonucleases; D008025:Ligases