Search

The polyanion heparin, whilst inhibiting free RNA polymerase, stimulates the rate of RNA synthesis in eucaryotic nuclei; it has been suggested that this process involves either the inactivation of RNase or the activation of blocked transcription complexes. These hypotheses have been investigated in isolated rat liver nuclei at 22 °C. We confirm that heparin inhibits free form A and B RNA polymerases but has little effect on the transcribing enzymes. The sensitivity of these enzymes to α-amanitin is unchanged by the agent and the stimulation is dependent on the presence of (NH4)2SO4 and Mn2+ ions. The size of the RNA product was analysed on denaturing formamide/ sucrose gradients. It shows only a small increase when heparin is included in the incubation. In addition, no significant size changes are observed if the incubation is continued in the presence of actinomycin D and α-amanitin which together inhibit 97% of all RNA synthesis. These results show that inactivation of RNase cannot be an explanation of the stimulatory effect in this system. Polynucleotide elongation rates and numbers of transcribing RNA polymerase molecules were determined by alkaline degradation of RNA labelled from [3H]UTP during l-min incubations. The [3H]UMP and [3H]uridine, derived from internal nucleotides and 3′-terminal nucleosides respectively, were separated by thin-layer chromatography and their tritium content measured. From this data the above parameters can be calculated. We show that the activity of RNase and phosphatase do not significantly affect the data and that levels of endogenous UTP are negligible. Data from eight separate experiments show that isolated rat liver nuclei contain 25-35 x 103 transcribing RNA polymerase molecules per diploid genome comprising 18-20 x 103 form A and 5- 15 x 103 form B. Form C was not detected.... [ABSTRACT FROM AUTHOR]

Recent findings have confirmed the role of form A DNA-dependent RNA polymerase activity as that which is responsible for the transcription of the ribosomal RNA-coding genes. Unfortunately, the form A enzymes have proved to be very labile and difficult to work with, especially under high ionic strength conditions. We have, therefore, investigated a method fot the purification of the form Al and All enzymes from rat liver using mild low-ionic-strength conditions. Since preparations from whole nuclei were found to be grossly contaminated with protein having similar properties, the enzymes are extracted from nucleoli. Forms Al and Al! are separated on a phosphocellulose column, purified by further ion-exchange chromatography, and by sedimentation through a glycerol gradient. The purified enzymes each migrate as a single band on native polyacrylamide gels and have the expected characteristics of form A RNA polymerase. Sedimentation rates through glycerol gradients indicate that they both have a similar size to that of Escherichia coil RNA polymerase (Mr, about 500000). The purified enzymes are free of DNase and RNase. A method is also described for the purification of form B from the nucleoplasm remaining after isolation of nucleoli. The presence of form C activity was not detected. [ABSTRACT FROM AUTHOR]

In many eucaryotic systems protein synthesis is coupled to ribosomal RNA synthesis such that shut-down of the former causes inhibition of the latter. We have investigated this stringency phenomenon in HeLa cells. The protein synthesis inhibitors cycloheximide and puromycin cause inactivation of both processes but valine starvation totally inhibits only the processing of 45-S RNA. DNA-dependent RNA polymerases form A, B and C (or I, II and III respectively) were extracted, separated partially by DEAE-cellulose chromatography and their activity levels determined. These do not decrease significantly during inhibition of protein synthesis. To find out whether or not form A is bound to its template under these conditions, proteins were removed from chromatin with the detergent sarkosyl. This does not affect bound RNA polymerase. Inhibition of protein synthesis caused up to 50% reduction in endogenous α-amanitin-insensitive chromatin-RNA-synthesising activity. This reduced level of activity was not affected by sarkosyl treatment. Levels in normal cells were stimulated. This result indicates that the form A RNA polymerase is not bound to its template when protein synthesis is inhibited. [ABSTRACT FROM AUTHOR]

Four of the six chromatin preparation methods examined here are shown to produce chromatins whose template properties differ radically from one another. Comparing the template-activity profiles obtainable with endogenous (i.e. chromatin-bound) and exogenous form B rat liver RNA polymerases across a range of salt concentrations, it appears that only one of these chromatins resembles isolated nuclei at all closely. A study of RNA product size on formamide gradients further reveals that only in this single case is there any sign of product-chains larger than 28-S rRNA. However, all these chromatins contain considerable exonucleotytic RNase activity, more marked at low than at high ionic strengths. In addition, prolonged (30-s) shearing is shown to cause drastic changes in the template properties of chromatin, as well as solubilizing it by reduction of fragment size. [ABSTRACT FROM AUTHOR]

The routine procedures used to prepare DNA from eucaryotic cells and nuclei give rise to material which is considerably degraded. This degradation takes the form of haplotomic (single-stranded) breaks arising from deoxyribonuclease action and diplotomic cleavage (double-stranded scission) of the DNA duplex due to mechanical shearing during the isolation procedure. Attempts to study the template specificities of the eucaryotic DNA-dependent RNA polymerases must take into account how these modifications to the structure of the DNA affect its template properties for these enzymes. Techniques are described which permit an accurate assessment of the state of integrity of the DNA. Treatment of the DNA with pancreatic deoxyribonuclease or sonication is shown to give rise to single-stranded interruptions in the duplex which are competent sites for the initiation of RNA synthesis by both forms AI and B rat liver RNA polymerases. However both these treatments also give rise to diplotomic cleavage of the DNA. This results in an inhibition of RNA synthesis which may be correlated with the formation of non-productive complexes by the RNA polymerases at DNA ends. Therefore the degree of template activation by pancreatic deoxyribonuclease and sonication for RNA synthesis as seen in the assay in vitro is a delicate balance between two opposing effects and is highly dependent upon the polymerase: DNA ratio. This data serves to emphasise that experiments designed to study the template specificity of eucaryotic RNA polymerases must make use of new, sophisticated techniques for the preparation of a template which is both intact and of high molecular weight. [ABSTRACT FROM AUTHOR]

DNA dependent RNA polymerase activity was extracted in a soluble form from rat liver nuclei by a mild, low salt, nonlytic technique. This technique preferentially extracts the form I polymerase which has been characterised by several laboratories. The enzyme was purified and separated into two species, present in approximately equal amounts, by DEAE-cellulose and phosphocelluose column chromatography. The two species, which have been designated form a and form Ib, were purified 40- and 100-fold, respectively, over the nuclear level with a recovery 0f 50-60%. Both forms have optimal activity at pH 8, sediment at 16 S in glycerol gradients, have a Km value for ATP of about 50 µM and are not inhibited by rifampicin or α-amanitin. No evidence could be obtained that the two forms represent a "core" and "complete" enzyme such as is produced by the bacterial enzyme on phosphocellulose. A standard competition hybridization technique could not distinguish between the RNA species produced by the two forms on a rat liver DNA template. [ABSTRACT FROM AUTHOR]

The transcription of rat-liver chromatin has been studied using partially purified form AI and highly purified form B DNA-dependent RNA polymerases isolated from rat liver. Chromatin contains endogenous RNA polymerase activity. This activity is evident only when the RNA polymerase assays are carried out at high ionic strength and its appearance as the ionic-strength increases is thought to be due to the removal of chromatin-associated proteins which block further transcription by the bound enzyme. This activity is insensitive to the rifampicin derivative AF/0-13 which is shown to inhibit initiation of RNA synthesis by mammalian RNA polymerases. Form AI polymerase is virtually inactive in the transcription of chromatin whereas the form B RNA polymerase (α-amanitin sensitive) actively transcribes chromatin. This activity has two salt concentration optima: (a) 0.01 M (NH4)2SO4 or 0.03 M KCl; (b) 0.16 M (NH4)2SO4 or 0.25 M KCl. Both these activities are inhibited by rifampicin AF/0-13. A comparison of the activity of the rat-liver form B polymerase and the Micrococcus lysodeikticus RNA polymerase demonstrates that chromatin is a more efficient template for the rat-liver enzyme. Evidence is presented that the rat-liver form B and the Micrococcus lysodeikticus RNA polymerases bind to and transcribe from different sites on the chromatin DNA. [ABSTRACT FROM AUTHOR]