Androgenic regulation of elongation of polyribonucleotide chains on rat ventral-prostate chromatin.

The kinetics of polyribonucleotide-chain elongation by rat ventral-prostate RNA polymerase B with homologous chromatin as a template were investigated. Chain elongation was measured under conditions wherein all initiation had occurred, no reinitiation took place and the reaction rate was constant. The kinetic behaviour of prostate RNA polymerase B was consistent with a mathematical model formulated for the multisubstrate enzyme. The addition of each nucleoside triphosphate was independent of the other three. The overall rate of chain elongation was lower when prostate chromatin from castrated rats was used than with prostate chromatin from normal rats. The inclusion of dihydrotestosterone-receptor complexes stimulated the rate of elongation. Androgenic effects did not appear to be directed towards the addition of individual nucleoside triphosphates, but probably towards one of the other major events in RNA-chain elongation, i.e., unwinding of DNA or movement of the enzyme along the template.     

Transcription of polyoma virus DNA after interaction with nuclear proteins in vitro.

Analysis of the nucleotide sequence at the 5′-triphosphate termini of RNA chains synthesized by T7 RNA polymerase from T7 DNA template indicates that nearly all RNA chains synthesized in this polymerase reaction contain the sequence, pppGpGp. In addition, studies carried out on T7 DNA-dependent ³²PP_i exchange into ribonucleoside triphosphates suggest that immediately following the guanine residues at the 5′-end of RNA formed in the T7 RNA polymerase reaction, there is one or more adenine residues. These results indicate a high degree of specificity of initiation of RNA synthesis by T7 RNA polymerase.     

Pre-4S RNA made in isolated HeLa cell nuclei terminates with U

Isolated HeLa cell nuclei are capable of synthesizing a precursor to 4S RNA. The 3′ terminus of the molecule made in vitro can be determined by four separate incubations with the four iritiated ribonucleoside triphosphates. The pre-4S was found to terminate specifically with uridine and be uniformly labeled. These facts are discussed in relation to specific termination of RNA molecules and possible reinitiation of RNA synthesis in isolated nuclei.     

RNA polymerase activity in bovine spermatozoa

Washed mature spermatozoa from bulls incorporate ribonucleoside triphosphates into RNA using an endogenous template. Maximum incorporation was observed at 31 degrees C in the presence of MgCl2, all four ribonucleoside triphosphates, beta-mercaptoethanol, and glycine sodium hydroxide buffer at pH 9.0. The amount of synthesis was linearly dependent upon the concentration of spermatozoa and continued for at least 4 h. Digestion studies revealed the RNA to be present in a protected (intracellular?) location in the spermatozoa. The RNA synthesis was inhibited by ethidium bromide, rifampicin, acriflavine, actinomycin D, and caffeine, but not by alpha-amanitine or rifamycin SV. Fractionation of the spermatozoa by sonication and separation of the heads and tails by centrifugation through a discontinuous gradient revealed that more than half of the total RNA polymerase activity was associated with the tail fraction.     

Chromatin- and nuclei-directed ribonucleic Acid synthesis in sugar beet root

The synthesis of RNA by chromatin-bound RNA polymerase prepared from sugar beet (Beta vulgaris) root tissue is completely dependent on the presence of a divalent metal (Mg²⁺ or Mn²⁺) and the presence of four ribonucleoside triphosphates. Accumulation of labeled acid-insoluble product is inhibited by the addition of RNase and actinomycin D to the reaction. When beet root slices are washed for 25 hours, chromatin-associated RNA polymerase activity increases 7-fold over that of unwashed tissue. This enzyme activity declines with further washing. DNA template availability, as measured by saturating levels of added Escherichia coli RNA polymerase, was also found to follow a pattern similar to that for RNA polymerase. Nearest neighbor frequencies of the RNA synthesized by chromatin isolated from unwashed and washed tissue are different.     

Effect of temperature on the transcription of T3 DNA b y T3-specific RNA polymerase in cell-free extracts of Escherichia coli CRT266

Cell free extracts were prepared from E. coli CRT266 9 min after infection with T3 phages. RNA synthesis in these extracts is almost entirely due to T3 RNA polymerase. The inactivation of T3 RNA polymerase in these extracts proceeds rapidly at 42 degrees C. 90% of the activity is lost within 10 min at this temperature. Under conditions where the formation of a stable initiation complex with T3 DNA is possible, i.e., in the presence of GPT, APT, and UTP the T3 RNA polymerase becomes protected against heat inactivation losing only )0% of its activity during an exposure to 42 degrees C for 10 min. Studies on the time course of RNA synthesis have shown that reinitiation is still possible at 37 degrees C and 42 degrees C. At 44 degrees C, however, RNA synthesis stops abruptly after 3 min indicating that reinitiation does no longer take place. The elongation of already initiated T3 RNA chains is rather resistant to heat. At 44 degrees C the same elongation rates are observed as at 37 degrees C and 42 degrees C, respectively.     

Isolation and properties of a testicular ribonucleic acid polymerase

A procedure is described for isolation in a soluble form of a ribonucleic acid polymerase from rat testes. Evidence is presented that this enzyme catalyzes three distinct reactions: (a) deoxyribonucleic acid-directed synthesis of RNA in the presence of all four major ribonucleoside triphosphates; (b) DNA-primed formation of polyadenylic acid and other ribohomopolymers in the presence of single ribonucleoside triphosphate substrates; (c) synthesis of complementary polyribonucleotides in the presence of various ribohomopolymer primers. The properties of these reactions are discussed with respect to metal ion requirements, affinities for ribonucleoside triphosphates and primer polynucleotides, heat denaturation of DNA primers, and the effects of ionic strength, beta-mercaptoethanol, polyamines, temperature, and inhibitors on the rates and extent of the reactions. The testicular ribonucleic acid polymerase is a very unstable enzyme that can be stabilized by high concentrations of glycerol.