Structural integrity of DNA and translational integrity of ribosomes in nuclease-treated cell-free protein synthesizing systems prepared from wheat germ and rabbit reticulocytes.
After treatment at a microsomal nuclease concentration too low to reduce the endogenous amino acid-incorporating activity of freshly prepared reticulocyte lysate, there is little, if any, intact 26 S RNA left in the ribosomes of either wheat germ or rabbit reticulocyte cell-free protein synthesizing extracts. The primary scissions, probably at highly exposed sites in the rRNA of plant and animal ribosomes, produce two fragments which remain complexed until thermal denaturation reveals "hidden breaks." Molecular weights of the fragments are approximately 0.5 x 10(6) and 0.8 x 10(6) in the case of wheat, and 0.4 x 10(6) and 1.3 x 10(6) in the case of rabbit. There is little perceptible degradation of 5 S, 5.8 S, and 18 S rRNA, or of tRNA in the same extracts. Even though limited degradation of 26 S rRNA by a reticulocyte nuclease has been reported to severely impair the translational mechanism in reticulocyte ribosomes, micrococcal nuclease-induced degradation of rRNA, whether limited or extensive, does not seriously impair the ability of reticulocyte lysates to discriminate, by selective polypeptide synthesis, between complex populations of cellular mRNA. In an allied study, it is shown that under conditions well suited to recovery of the 5.8 S/26 S rRNA complex, with its naturally occurring hidden break, 5 S/18 S rRNA complexing is not detectable in the RNA or metabolizing embryos, nor in the RNA from untreated or nuclease-treated protein synthesizing extracts from wheat and rabbit. The significance of this finding is briefly elaborated in relation to the suggestion that 5 S rRNA may interact with the M2(6)A-m2(6)A hairpin near the 3'-end of 18 S rRNA.
D000818:Animals; D002474:Cell-Free System; D008836:Micrococcal Nuclease / Q000494:pharmacology; D008970:Molecular Weight; D010944:Plants / Q000378:metabolism*; D014176:Protein Biosynthesis / Q000187:drug effects; D012335:RNA, Ribosomal / Q000235:genetics*; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism*; D012270:Ribosomes / Q000187:drug effects / Q000378:metabolism*; D014908:Triticum / Q000378:metabolism
D012335:RNA, Ribosomal; D008836:Micrococcal Nuclease