Isolation and properties of highly purified Halobacterium cutirubrum deoxyribonucleic acid-dependent ribonucleic acid polumerase

Abstract

1. The subunits alpha and beta of Halobacterium cutirubrum DNA-dependent RNA polymerase have been purified to electrophoretic homogeneity. Both have mol.wt. 18000 and they are required in equimolar amounts for optimum activity. 2. The instability of the complete enzyme, alphabeta, in the absence of salt is due to the rapid inactivation of the beta subunit in these conditions. 3. Nearest-neighbour analysis of the product formed on poly[d(A-T)] as template shows that the enzyme copies the latter accurately. 4. The enzyme initiates new chains with purine nucleoside triphosphates exclusively. 5. The product obtained in the standard assay conditions contains some high mol.wt. (>16S) material, but consists primarily of short chains, of average length 70-80 nucleotide units. 6. The template specificity of the complete enzyme has been studied at high and low ionic strength. Its extreme dependence on salt concentration is unrelated to the gross overall base composition of the DNA used. 7. T(7) DNA is transcribed asymmetrically and the enzyme selectively copies the T(7) ;early' genes. 8. Preliminary amino acid analyses of alpha and beta subunits show that their overall content of acidic, basic and neutral amino acids does not differ appreciably from that of Escherichia coli RNA polymerase.

Purification and properties of the ribonucleic acid-dependent ribonucleic acid polymerase from Halobacterium cutirubrum

“…ofbacteria and the pH4 precipitation was performed as described by Louis & Fitt (1972a). The pellet after acid precipitation was suspended in the minimum volume of high-salt buffer and was dialysed against the latter (200vol.)…”

“…Solid (NH4)2SO4 was then added to 80% saturation; the precipitate was removed by centrifugation and was discarded, and the supernatant was dialysed overnight against high-salt buffer. The resulting solution (20-30ml) was passed at 40ml/h by upward flow through a column (5cm x 85cm) of Bio-Gel P-60 prepared, stabilized and operated as described by Louis & Fitt (1972a). The combined active fractions (100-130ml) were concentrated to approx.…”

“…H. cutirubrum RNA-dependent RNA polymerase activity was measured by the standard assay (Louis & Fitt, 1971b, 1972a. The assay medium (0.1 ml) contained: tris(hydroxymethyl)-methylaminopropanesulphonic acid-NaOH buffer, pH9.5, 10,umol; high-molecular-weight RNA, 75,tg; [14C]ATP, Snmol (about 10000 d.p.m./nmol); CTP,GTP and UTP,20nmol each;MgCl2,10,umol;MnCl2,ltmol;KCI,150,umol;NaCl,60,mol; enzyme, up to 3 units.…”

“…cutirubrum DNA-dependent RNA polymerase was assayed as described by Louis & Fitt (1972a 10,umol;MnC12,1,umol;KCI,150,uimol;NaCl,60,umol; (Louis & Fitt, 1971b) we used tris-HCl buffer, pH9.5, in the standard assay of the RNAdependent enzyme. Recently, for a period of several months, we obtained high blanks with tris buffers, owing apparently to the presence in the public water supply of a steam-distillable oxidizing agent that caused rapid oxidation of Mn21 in the presence of tris buffer.…”

“…Micro-disc electrophoresis (Neuhoff, 1968) in 8, 10 and 12% (w/v) polyacrylamide gels at pH 7.0, 8.0 or 9.4 was performed as described by Louis & Fitt (1972a).…”