A mammalian cell-free transcriptional system was developed in which mammalian RNA polymerase synthesizes globin messenger RNA sequences from bone-marrow chromatin. The messenger RNA sequences are detected by measurement of the ability of the transcribed RNA to hybridize with globin complementary DNA. The globin complementary DNA is synthesized by the enzyme from avian myeloblastosis virus, RNA-directed DNA polymerase, with purified globin messenger RNA as template. The specificity of the globin complementary DNA in annealing reactions was verified by preparing DNA complementary to liver messenger RNA and showing that the globin and liver complementary DNAs are specific for their own messenger RNAs. Both DNA-dependent RNA polymerase II from sheep liver and RNA polymerase from Escherichia coli can transcribe globin messenger RNA sequences from rabbit bone-marrow chromatin; however, the mammalian enzyme appears to be more specific in that globin gene sequences represent a higher proportion of the RNA synthesized. Neither polymerase can transcribe globin messenger RNA sequences from rabbit-liver chromatin. This cell-free assay system should be useful in searching for mammalian transcriptional regulatory factors.

The synthesis of DNA complementary to rabbit globin mRNA by the enzyme RNA-directed DNA polymerase (from avian myeloblastosis virus) has been studied. The reaction is dependent on mRNA as template, oligo(dT) as primer, and the four deoxynucleoside triphosphates as substrates. The 28 S, 18 S, and 4 S RNA are not effective templates; oligo(dG), oligo(dC), and oligo(dA) are not effective primers. The product of the reaction is a DNA-RNA hybrid, the DNA of which has an electrophoretic mobility of 7 to 10 S. The DNA hybridizes with globin 10 S mRNA but not with 28 S, 18 S, or 4 S RNA, nor does it hybridize with an 8 to 12 S RNA fraction isolated from rabbit liver polysomes. Thus, the RNA-directed DNA polymerase is capable of synthesizing a DNA molecule complementary to globin mRNA; however, based on its size the product does not appear to be a complete copy of the RNA template. Using rabbit reticulocyte 10 S globin mRNA or rabbit liver mRNA as template the RNA-directed DNA polymerase can synthesize poly(dT) when dTTP is the only substrate present; no reaction occurs in the presence of any other single deoxynucleoside triphosphate. A high molecular weight poly(dT) polymer is formed, presumably transcribed from the poly(A) region of the mRNA. Thus, under some conditions, the enzyme may slip during transcription so that parts of the RNA are transcribed more than once in the synthesis of a single DNA molecule.

This chapter aims to develop a method for the preparation and purification of active ribosomal subunits from rat and mouse liver. The technique involves incubating purified polysomes with all components for protein synthesis until all competent ribosomes terminate and release their polypeptide chain. Upon treatment such an incubation mixture, all ribosomes are found as 60 S and 40 S subunits. The purified subunits, when recombined, spontaneously associate to 80 S couples in the absence of tRNA, mRNA, or supernatant factors. The purified 60 S subunit contains the enzymatic site for the catalysis of peptide bond formation. Purified mouse liver 60 S subunits are able to catalyze the formation of peptide bonds using substrates puromycin and N-formyl- or N-acetylaminoacyl oligonucleotides from tRNA. A relative comparison of the rates of N-acetylleucyl-puromycin formation catalyzed by E. coli 50 S subunits and mouse liver 60 S subunits shows that the reaction rate with E. coli ribosomes is approximately twice that with liver subunits.

1.(1) During in vitro polypeptide chain termination by rat or mouse liver polysomes, 80 s monosomes briefly dissociate into free subunits, presumably at the moment of their relase from the messenger. The subunits reassociate to form 80 s couples. In this reassociation step labeled 40 s subunits added to the incubation mixture freely compete with unlabeled subunits.2.(2) The 80 s ribosomes reformed after termination of the polypeptide chain slowly exchange their subunits with radioactive subunits in the reaction mixture. The equilibrium between free subunits and 80 s couples strongly favors the latter.3.(3) In contrast to polypeptide chain termination, at initiation of polyphenylalanine synthesis directed by polyuridylic acid, 80 s ribosomes attach to this messenger without a dissociation step.

We have developed a method for preparation and purification of active ribosomal subunits from rat and mouse liver. Our technique involves incubating purified polysomes with all components for protein synthesis until all competent ribosomes have terminated and released their polypeptide chain. Upon treatment of such an incubation mixture with 0.5 m-KCl, followed by sucrose gradient centrifugation in 0.3 m-KCl and 0.002 or 0.003 m-magnesium acetate, 80 to 90% of all ribosomes are found as 60 s and 40 s subunits. These subunits can be concentrated from the gradient fractions by ethanol precipitation. The purified subunits, when recombined, spontaneously associate to 80 s couples in the absence of transfer RNA, messenger RNA or supernatant factors. When supplied with poly U and the other components required for in vitro polypeptide synthesis, the subunits polymerize 15 to 20 phenylalanine residues per subunit couple present in the reaction mixture. Furthermore, at least 50 to 65% of the subunits actually participate in polyphenylalanine synthesis. At least 90% of the RNA of the purified subunits is intact as shown by sedimentation analysis of lithium dodecyl sulfate-treated particles. The purified 60 s subunit contains the enzymic site for the catalysis of peptide bond formation. This reaction is sensitive to anisomycin, an inhibitor of mammalian protein synthesis, but is not affected by the bacterial inhibitor chloramphenicol nor by cyclohexamide, an inhibitor of translocation in mammalian systems. The proteins from 40 s and 60 s subunits and from polysomes were analyzed and compared by acrylamide gel electrophoresis.