Isoproterenol-induced selective phosphorylation invivo of the 214,000 dalton subunit of rat C6 glioma cell RNA polymerase II

The phosphorylation $\text{in}\text{vivo}$ of RNA polymerase II after isoproterenol stimulation of confluent rat C6 glioma cell cultures has been investigated. Glioma cells were incubated in the presence of Na₂H³²PO₄ and stimulated for 1 hour with the β-adrenergic agonist isoproterenol. The phosphorylation pattern was analyzed after purification of RNA polymerase II by immunoprecipitation, SDS-polyacrylamide gel electrophoresis and autoradiography. Isoproterenol markedly increased [³²P]phosphate incorporation into the 214,000 dalton RNA polymerase subunit. Analysis of the phosphate acceptor amino acid revealed the presence of only [³²P]phosphoserine. The data demonstrates an isoproterenol-induced structural modification of RNA polymerase II.     

Differential effect of neomycin on DNA dependent -DNA and RNA synthesis in vitro

Neomycin inhibits $\text{in vitro}$ DNA dependent DNA and RNA synthesis catalyzed by DNA polymerase I and RNA polymerase from $\text{E. coli}$. The effect of the antibiotic is more pronounced towards DNA synthesis. The inhibition of DNA synthesis is competitive with template DNA, does not reverse with excess deoxynucleoside triphosphate, Mg²⁺ or enzyme $\text{E. coli}$ DNA polymerase I. Neomycin does not reduce the number of potential 3′ -OH end or primer. It seems to shorten the size of the newly formed polynucleotide.     

Antigenic homology of eukaryotic RNA polymerases

Facilitated by an improved enzyme purification procedure, antisera to calf thymus DNA-dependent RNA polymerase II was prepared in hens. Using immunoprecipitation and inhibition of enzymatic activity the immunological properties of several eukaryotic RNA polymerases were examined. Purified calf thymus and rat liver polymerase II exhibited antigenic homology. The partially purified amphibian ($\text{Xenopus laevis}$) and protozoan ($\text{Tetrahymena pyriformis}$) polymerase II had reduced crossreactivities. Calf thymus polymerase I also shared antigenic homology with the form II enzymes.     

Viral nucleocapsid melting to measure protein: RNA interactions

Thermal denaturation of nucleocapsids of wild type (WT) vesicular stomatitis virus (VSV), containing only the nucleocapsid protein (N) and viral RNA, caused a “melting” that resulted in an A_260nm absorbance increase of 140%. The nucleocapsids of two temperature-sensitive ($\text{ts}$) VSV mutants, $\text{ts}$ G31BP and $\text{ts}$ G22, both underwent larger absorbance increases of 251% and 177% respectively, suggesting these nucleocapsids are complexed by weaker N protein: RNA interactions than the WT-VSV. Two other mutants, $\text{ts}$ G31 and $\text{ts}$ G41 underwent A_260nm increases either similar to, or smaller than, that measured with WT-VSV nucleocapsids. RNA synthesis by $\text{ts}$ G31BP in infected cells was also found to be decreased at elevated temperatures. This temperature sensitive defect in viral RNA metabolism in $\text{ts}$ G31BP may be the result of weaker protein:RNA interactions associated with the nucleocapsid.     

5' Cap methylation of homologous poly A(+) RNA by a RNA (guanine-7) methyltransferase from Neurospora crassa.

RNA (guanine-7) methyltransferase, partially purified from $\text{N.}\text{crassa}$ mycelia, catalyzed the transfer of the methyl group from S-adenosylmethionine to the 5′ terminus of both $\text{N.}\text{crassa}$ poly A(+) RNA and reovirus unmethylated mRNA. RNase T₂ digestion of the $\text{in}\text{vitro}$ methylated poly A(+) RNA from $\text{N.}\text{crassa}$ yielded the “cap” structures m ⁷G(5′)pppAp and m ⁷G(5′)pppGp in a ratio of 2:1 respectively. RNase T₂ digestion of the $\text{in}\text{vitro}$ methylated reovirus mRNA yielded m ⁷G(5′)pppGp exclusively. The absence of mRNA 2′-0-methyltransferase activity in the enzyme preparation is consistent with the absence of 2′-0-methylation in $\text{N.}\text{crassa}$ mRNA [Seidel, B. L. and Somberg, E. W. (1978) Arch. Biochem. Biophys. $\text{187}$, 108–112]. This is the first isolation of an eucaryotic, cellular RNA (guanine-7) methyltransferase that has been shown to methylate homologous substrate.     

Synthesis of RNA polymerase in Escherichia coli B-r growing at different rates

Polyacrylamide gel electrophoresis of unfractionated sodium dodecyl sulfate lysates of Escherichia coli$\text{B}\text{r}$ has been used to investigate the synthesis of β and β′ subunits of RNA polymerase as a function of bacterial growth rate. In succinate (μ = 0.67 doublings/h), glucose (μ = 1.36 doublings/h) and glucose/amino acids (μ = 2.10 doublings/h) supplemented media, the fraction of [¹⁴C]leucine-labeled β and β′ protein/total protein was found to be 1.05, 1.31 and 1.56%, respectively. Comparison of these values with recent estimates from this laboratory of the differential rate of synthesis of functioning RNA polymerase suggests an excess of total over functioning RNA polymerase. The significance of these data in reference to the regulation of RNA polymerase synthesis is discussed.     

The early synthesis and possible function of a 0.5 X 10(6) Mr RNA after transfer of dark-grown Spirodela plants to light.

A 0.5 × 10⁶$\text{M}$_r RNA found in plastids of the aquatic angiosperm $\text{Spirodela}$, is synthesized at a much higher rate than any other rapidly labeling RNA species about $\text{3–3}\text{1}\text{2}$ h after dark-grown plants are transferred to light. The pulse labeling kinetics of the 0.5 × 10⁶$\text{M}$_r RNA after transfer to light, argue against its involvement in the biogenesis of plant rRNAs. Although poly(A) RNA is found in $\text{Spirodela}$, poly(A) sequences are not detected in the 0.5 × 10⁶$\text{M}$_r RNA; yet a sucrose gradient fraction which includes RNA of this $\text{M}$_r stimulates amino acid incorporation by an $\text{E. coli}$ cell free extract more than other RNA fractions. The possible involvement of the 0.5 × 10⁶$\text{M}$_r RNA as a chloroplast messenger is discussed.     

Methylation of transfer RNA during transformation of human lymphocytes by phytohemagglutinin

Methylation of transfer RNA during phytohemagglutinin induced transformation of human lymphocytes was studied by labeling the tRNA $\text{in}\text{vivo}$ with (methyl-H³)-methionine and measuring the distribution of tritium in the methylated nucleotides. An alteration in the pattern of methylation occurred within hours after PHA-stimulation and this change was maintained through several cell generations. There was a 50 to 94% increase in the relative amount of methylated N²-methyl-guanine (1 to 3 hr) and a 40 to 59% decrease in the relative amount of 1-methyladenine (1 to 12 hr). Treatment of the stimulated cells with Actinomycin D prevented the subsequent methylation not tRNA. However, inhibition of protein synthesis by puromycin did not effect the early changes observed in the methylation of tRNA.     

Electron microscopic studies of the binding of Escherichia coli RNA polymerase to DNA: II. Formation of multiple promoter-like complexes at non-promoter sites

The interactions between Escherichia coli RNA polymerase holoenzyme and a 3800 base-pair restriction fragment of bacteriophage T7 DNA (Mbo-IC) have been examined by electron microscopy. In addition to exhibiting weak, non-specific interactions (K_a ～ 10⁴ M^?1), RNA polymerase is able to form up to 15 to 20 relatively stable complexes with this template (K_a est 10⁹ M^?1). Only one of these complexes is formed at the T7 promoter E, that maps at 92.2 ± 1% on the conventionalgenome. The remaining complexes seem to be situated non-randomly on this fragment and possibly involve interactions with specific DNA sequences. The association kinetics of formation have been examined and give rise to a second-order rate constant of ～ 10⁵ M^?1s^?1. Formation of these complexes is markedly reduced at low temperatures. Under standard binding conditions (50 mM-NaCl) the dissociation rate of these complexes is slow ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext><mtext>\; ~\; 30\; </mtext><mtext>min</mtext>}}$), but increases rapidly with increasing salt concentration and at reduced temperatures. It is unaffected by the presence of heparin up to 5 μg/ml. Thus it appears that E. coli RNA polymerase can form complexes with promoter-like properties at many different sites on T7 DNA.     

1,10-Phenanthroline-cuprous ion complex, a potent inhibitor of DNA and RNA polymerases.

The inhibition by 1,10-phenanthroline of $\text{E. coli}$ DNA polymerase I has recently been attributed to the formation in the assay mixtures of a unique and effective inhibitor, the 2:1 1,10-phenanthroline-cuprous ion complex (1). We have now found that this coordination complex is also an effective inhibitor of $\text{E. coli}$ DNA dependent RNA polymerase, Micrococcus luteus DNA dependent DNA polymerase, and T-4 DNA dependent DNA polymerase. This conclusion is based either on the requirement of a thiol for 1,10-phenanthroline inhibition or on the reversal of 1,10-phenanthroline inhibition by the non-inhibitory cuprous ion specific chelating agent 2,9-dimethyl-1,10-phenanthroline. 2,2′,2″-Terpyridine is also very effective at relieving 1,10-phenanthroline inhibition. The reversal of 1,10-phenanthroline inhibition should be attempted before it is claimed that 1,10-phenanthroline inhibits any polymerases by coordinating a zinc ion at the active site.     

Effects of 3′-deoxyadenosine triphosphate on in vitro RNA synthesis of plant RNA-dependent RNA polymerases

3′-deoxyadenosine triphosphate inhibited $\text{in}\text{vitro}$ [³H]UMP incorporation by RNA-dependent RNA polymerases from tobacco and cowpea plants. The inhibition of [³H]UMP incorporation could be reversed by simultaneous addition of higher ATP concentrations but not with increasing concentrations of UTP or when excess ATP was added 10 min after the inhibitor. These results suggest 3′-deoxyadenosine triphosphate competes specifically with ATP in reaction mixtures and results in premature termination of RNA synthesis $\text{in}\text{vitro}$ by RNA-dependent RNA polymerase.