Multiple forms of DNA-dependent RNA and polyadenylic acid polymerases from heterotrophically grown Rhodospirillum rubrum.

Three, two major and one minor, distinct RNA polymerases have been isolated and partially purified from heterotrophically grown Rhodospirillum rubrum, a facultative photosynethetic bacterium. Associated with each of these three enzymes is a distinct polyadenylic acid polyemrase. All of these enzyme activities are dependent on DNA templates and are resistant to rifampicin and streptovaricin. The structural subunit composition, the response to various chemical compounds and DNA templates, and the properties of the products of these enzymes are studied in detail and compared with those of similar enzyme activities from other bacterial systems. Several unique features have been observed in the R. rubrum enzyme systems, such as an uneven incorporation of purine and pyrimidine nucleotides by the RNA polymerases, and the presence of a lag period in the polyadenylic acid polymerase activities.     

Multiple forms of DNA-dependent DNA polymerase during early development and in somatic cells of Xenopus laevis.

Four distinct DNA-dependent DNA polymerase activities (DNA polymerases I, II, III and IV according to the order of elution from a DEAE column) have been separated from extracts of unfertilized Xenopus laevis eggs. The same activities, on the basis of their chromatographic properties, template specificities and sedimentation coefficients, have been found in embryos at least until the gastrula stage. On the other hand, Xenopus kidney cells grown in culture, as well as full grown oocytes lack DNA polymerase I. These data suggest the DNA polymerase I might be a special DNA polymerase activity involved in the extremely rapid DNA synthesis which takes place during early development of X. laevis.     

Synthesis and properties of fluorescent nucleotide substrates for DNA-dependent RNA polymerases.

A new class of fluorescent nucleotide analogs which contain the fluorophore 1-aminonaphthalene-5-sulfonate attached via a gamma-phosphoamidate bond has been synthesized. Both the purine and pyrimidine analogs have fluorescence emission maxima at 460 nm. Cleavage of the alpha-beta-phosphoryl bond produces change in both the absorption and fluorescence emission spectra. The fluorescence of the pyrimidine analogs is quenched; cleavage of the alpha-beta-phosphoryl bond of the UTP analog produces about a 14-fold increase in fluorescence intensity at 500 nm. Under the same conditions the fluorescence of the CTP analog increases about 8-fold, whereas the fluorescence of the purine analogs shows only a slight change. These derivatives are good substrates for Escherichia coli RNA polymerase with only slightly increased Km values and with Vmax values about 50 to 70% that of the normal nucleotides. They are used less efficiently by wheat germ RNA polymerase II. The ATP analog can be used by E. coli RNA polymerase to initiate RNA chains.     

Animal DNA-dependent RNA polymerases. Purification and molecular structure of hen-oviduct and liver class-B RNA polymerases.

Hen ovidcut and liver class B RNA polymerases have been extensively purified and their molecular structure has been analysed. While only one enzyme B form (BIIb) was found in liver, three forms (BI, BIIa and BIIb) were resolved from oviduct. The molecular structures of the various class B RNA polymerase forms purified from hen oviduct and liver are identical to the corresponding forms previously purified from calf thymus and rat liver. At the present level of resolution the only difference lies in a slight difference in the charge of one subunit (SB2a) of enzyme form BIIa, when comparing the mammal and bird enzymes. It is unlikely that the absence of enzyme forms BI and BIIa in purified hen liver RNA polymerase B could be related to limited and specific proteolysis during the purification, since co-purification of oviduct and liver RNA polymerase B activities from a mixture of oviduct and liver nuclei does not affect the presence of either oviduct enzyme form BI or BIIa in the final purified mixture.     

Early evolution of eukaryotic DNA-dependent RNA polymerases

Eukaryotic DNA-dependent RNA polymerases (Pol I-III) share a conserved core of 12 subunits, which is closely related to archaeal RNA polymerases. Rpb8, a subunit found in Pol I, II and III, was thought to be restricted to eukaryotes. We show here that Rpb8 closely resembles an archaeal protein called G, found only in Crenarchaea, which identifies a last missing link between the core structure of archaeal and eukaryotic RNA polymerases.     

Synthesis of two DNA-dependent RNA polymerases in yeast

Specific activities of Saccharomyces cerevisiae RNA polymerases I and II were measured in cells growing under different nutrient conditions and throughout the mitotic cell cycle. The specific activity of RNA polymerase I (possibly the ribosomal polymerase) does not vary during the yeast cell cycle. In contrast the specific activity of RNA polymerase II (messenger polymerase) increases during the first third of the cycle and thereafter declines. The independent regulation of synthesis of these two enzymes is further emphasised by observations on the response to different nutrient conditions. Shifting cells from minimal to rich medium led to enhanced RNA polymerase I activity but very little change in activity of RNA polymerase II. Furthermore the activity of RNA polymerase I varies directly with change in growth rate whereas the activity of RNA polymerase II is approximately constant over a range of growth rates. From this data it is suggested: (i) The synthesis of these two enzymes is independently regulated; (ii) RNA polymerase I is synthesised continuously throughout the cycle whereas RNA polymerase II is synthesised periodically early in the cell cycle.     

Nuclear DNA-dependent RNA polymerases of Ehrlich ascites tumor cells: two discrete -amanitin-sensitive forms

DNA-dependent RNA polymerase was solubilized from nuclei of ascites tumor cells by the standard techniques of ultrasonic treatment in 0.3 M ammonium sulfate, salt fractionation, and dialysis. Three discrete forms of RNA polymerase (I, II, III) were separated on DEAE-Sephadex A-25. Forms II and III were inhibited by α-amanitin, but no form was sensitive to rifampicin. Each form was more active with Mn⁺⁺ than with Mg⁺⁺ ions, more active with denatured than with native calf thymus DNA, and differed from the others with respect to optimal concentrations of (NH₄)₂SO₄, Mn⁺⁺ ions and DNA.     

Structural homology between different archaebacterial DNA-dependent RNA polymerases analyzed by immunological comparison of their components

The archaebacterial DNA-dependent RNA polymerases have a complex structure containing eight or more components. Immunochemical analysis shows an extensive homology between the components of the enzymes of nine different species. Two enzyme subtypes can be distinguished: that of the thermoacidophilic and/or sulfur-metabolizing archaebacteria with the composition BACDEFGHIJ and that of the methanogenic plus halophilic archaebacteria with the composition ABB'C(D).... Components B and B' of the latter subtype probably evolved by the division of the large component B of the BACD... type enzyme. The existence of the two subtypes corroborates the division of the archaebacteria into two phylogenetic main branches.