Trinucleotide AUA and UAU formation catalyzed by wheat germ RNA polymerase II

1989 ◽  
Vol 54 (3) ◽  
pp. 811-818 ◽  
Author(s):  
Aleš Cvekl ◽  
Květa Horská ◽  
Karel Šebesta ◽  
Ivan Rosenberg ◽  
Antonín Holý
Keyword(s):  
Ionic Strength ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Wheat Germ ◽  
Catalytic Mechanism ◽  
Bond Formation ◽  
E Coli ◽  
Substrate Properties ◽  
Rna Polymerase Holoenzyme ◽  
Analogous Mechanism

The elongation of dinucleotides ApU and UpA to trinucleotides ApUpA and UpApU by wheat germ RNA polymerase II was studied at a medium ionic strength (60 mM-KCl). The catalytic mechanism of the first internucleotide bond formation consists in the binding of the primer dinucleotide followed by the binding of NTP ("ordered bibi" reaction), i.e. by an analogous mechanism as found for RNA polymerase holoenzyme from E. coli. In further experiments phosphonate analogues of dinucleotides ApU and UpA were used as the priming dinucleotides. It was shown that analogues U(c)pA and Up(c)A are very poor primers for the synthesis of corresponding trinucleotides; the elongation of analogues A(c)pU and Ap(c)U was not observed at all. The comparison of kinetic constants Kia, KmA, KmB and Vmax as well as the substrate properties of phosphonate analogues indicates the increased specifity of the wheat germ RNA polymerase initiation binding site in comparison with the E. coli holoenzyme.

Cibacron Blue inhibition of prokaryotic and eukaryotic DNA-dependent RNA polymerases

1990 ◽  
Vol 55 (11) ◽  
pp. 2769-2780
Author(s):  
Aleš Cvekl ◽  
Květa Horská
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Wheat Germ ◽  
Rna Polymerase Iii ◽  
Enzymic Activity ◽  
Rna Polymerases ◽  
Cibacron Blue ◽  
E Coli ◽  
Polymer Formation ◽  
Calf Thymus

A comparison was drawn between the action of Cibacron Blue F3GA on the enzymic activity of DNA-dependent RNA polymerases from different sources, e.g. Escherichia coli, calf thymus and wheat germ (polymerase II). Sensitivity towards this inhibitor was determined for polymer formation and primed abortive synthesis of trinucleotide UpApU. In case of E. coli polymerase and wheat germ polymerase II the dye inhibits both polymer formation and abortive synthesis. Calf thymus polymerase II is inhibited only in the polymerisation step. The primed initiation reaction was found to be resistant towards the dye. In case of E. coli polymerase and wheat germ polymerase II the sensitive step is the formation of internucleotide bond whereas in case of calf thymus polymerase II the translocation of the enzyme is influenced. An analysis of kinetic data indicates more than one binding site for the dye on RNA polymerase II from calf thymus and wheat germ. Cibacron blue does not inhibit specific transcription catalyzed by RNA polymerase III from human HeLa cells and mouse leukemia L1210 cells.

scholarly journals Studies on sex-organ development. Changes in the oestrogenic response of the chick Müllerian duct as measured by chromatin template and ribonucleic acid initiation capacity

1979 ◽  
Vol 182 (2) ◽  
pp. 257-269 ◽  
Author(s):  
G K Andrews ◽  
C S Teng
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Wheat Germ ◽  
Average Rate ◽  
Developmental Trend ◽  
Mullerian Duct ◽  
Müllerian Duct ◽  
E Coli ◽  
Chromatin Template

Assays of transcription in vitro, with Escherichia coli RNA polymerase or wheat-germ RNA polymerase II, were used to characterize chromatin templates isolated from the left Müllerian duct of the chick embryo during normal development, and during development in the presence of diethylstilboestrol. Control Müllerian-duct template capacity with E. coli RNA polymerase decreased from 6.42% on day 10 to 4.34% by day 15 of development. Similar results were found with wheat-germ RNA polymerase II. In the presence of rifampicin and heparin, the prokaryotic enzyme transcribed a number-average RNA chain of 670 nucleotide residues, at an average rate of 110 nucleotide residues/min, from Müllerian-duct chromatin of all developmental stages. From day 10 to day 15 there was a 44% decrease in the number of initiation sites for E. coli RNA polymerase on Müllerian-duct chromatin. A 47% decline was observed when these chromatins were transcribed with excess RNA polymerase II in the presence of rifamycin Af/013. Signs of increasing responsiveness to oestrogen developed between days 10 and 16. Embryos exposed to maximally responsive doses of diethylstilboestrol for 2 days showed increases in Müllerian-duct chromatin template capacity, RNA-chain initiation sites, wet weight, protein and RNA. The changes seen in the oviduct of the 1-week-old chick injected for 2 days with diethylstilboestrol were defined as 100% responses. By comparison, the Müllerian duct, after exposure to diethylstilboestrol from day 10 to day 12, from day 13 to day 15 or from day 16 to day 18, showed a 15%, 39% and 72% template response respectively, and a 42%, 56% and 85% initiation-site change respectively. A similar developmental trend was observed in all parameters. It is concluded that oestrogenic responsiveness in the developing Müllerian duct increases from day 10 to nearly maximal values by day 16 of development, and that this transition is paralleled by a progressive restriction of genomic activity.

scholarly journals Studies on sex-organ development. Changes in chromatin structure during spermatogenesis in maturing rooster testis as demonstrated by the initiation pattern of ribonucleic acid synthesis in vitro

1978 ◽  
Vol 170 (2) ◽  
pp. 203-210 ◽  
Author(s):  
C Mezquita ◽  
C S Teng
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Wheat Germ ◽  
Acid Synthesis ◽  
Chromatin Interaction ◽  
E Coli ◽  
Double Stranded Dna ◽  
The Stability ◽  
Kinetics Of

To probe the structural change in the genome of the differentiating germ cell of the maturing rooster testis, the chromatin from nuclei at various stages of differentiation were transcribed with prokaryotic RNA polymerase from Escherichia coli or with eukaryotic RNA polymerase II from wheat germ. The transcription was performed under conditions of blockage of RNA chain reinitiation in vitro with rifampicin or rifampicin AF/013. With the E. coli enzyme, the changes in (1) the titration curve for the enzyme-chromatin interaction, (2) the number of initiation sites, (3) the rate of elongation of RNA chains, and (4) the kinetics of the formation of stable initiation complexes revealed the unmasking of DNA in elongated spermatids and the masking of DNA in spermatozoa. In both cases the stability of the DNA duplex in the initiation region for RNA synthesis greatly increased. In contrast with the E. coli enzyme, the wheat-germ RNA polymerase II was relatively inefficient at transcribing chromatin of elongated spermatids. Such behaviour can be predicted if unmasked double-stranded DNA is present in elongated spermatids.

scholarly journals Functional dissection of the catalytic mechanism of mammalian RNA polymerase II

2005 ◽  
Vol 83 (4) ◽  
pp. 497-504 ◽  
Author(s):  
Benoit Coulombe ◽  
Marie-France Langelier
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Catalytic Mechanism ◽  
Mutational Analysis ◽  
Structural Elements ◽  
Catalytic Mechanisms ◽  
Rnap Ii ◽  
Affinity Peptide

High resolution X-ray crystal structures of multisubunit RNA polymerases (RNAP) have contributed to our understanding of transcriptional mechanisms. They also provided a powerful guide for the design of experiments aimed at further characterizing the molecular stages of the transcription reaction. Our laboratory used tandem-affinity peptide purification in native conditions to isolate human RNAP II variants that had site-specific mutations in structural elements located strategically within the enzyme's catalytic center. Both in vitro and in vivo analyses of these mutants revealed novel features of the catalytic mechanisms involving this enzyme.Key words: RNA polymerase II, transcriptional mechanisms, mutational analysis, mRNA synthesis.

scholarly journals A DNA-dependent RNA synthesis by wheat-germ RNA polymerase II insensitive to the fungal toxin α-amanitin

1992 ◽  
Vol 285 (1) ◽  
pp. 85-90 ◽  
Author(s):  
C Job ◽  
D Shire ◽  
V Sure ◽  
D Job
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Wheat Germ ◽  
Rna Synthesis ◽  
Biochemical Properties ◽  
Size Analysis ◽  
Reaction Products ◽  
High Concentration ◽  
Fungal Toxin ◽  
Alpha Amanitin

Wheat-germ RNA polymerase II is able to catalyse a DNA-dependent reaction of RNA synthesis in the presence of a high concentration (1 mg/ml) of the fungal toxin alpha-amanitin. This anomalous reaction is specifically directed by single-stranded or double-stranded homopolymer templates, such as poly(dC) or poly(dC).poly(dG), and occurs in the presence of either Mn2+ or Mg2+ as the bivalent metal cofactor. In contrast, the transcription of other synthetic templates, such as poly(dT), poly(dA).poly(dT) or poly[d(A-T)] is completely abolished in the presence of 1 microgram of alpha-amanitin/ml, in agreement with well-established biochemical properties of class II RNA polymerases. Size analysis of reaction products resulting from transcription of (dC)n templates of defined lengths suggests that polymerization of RNA chains proceeds through a slippage mechanism. The fact that alpha-amanitin does not impede this synthetic reaction implies that the amatoxin interferes with the translocation of wheat-germ RNA polymerase II along the DNA template.

scholarly journals Effect of salts on abortive and productive elongation catalysed by wheat germ RNA polymerase II

1986 ◽  
Vol 14 (4) ◽  
pp. 1583-1597 ◽  
Author(s):  
Jacques Dietrich ◽  
Marcel Teissere ◽  
Claudette Job ◽  
Dominique Job
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Wheat Germ ◽  
Productive Elongation

scholarly journals Structure of GPN-Loop GTPase Npa3 and Implications for RNA Polymerase II Assembly

2015 ◽  
Vol 36 (5) ◽  
pp. 820-831 ◽  
Author(s):  
Jürgen Niesser ◽  
Felix R. Wagner ◽  
Dirk Kostrewa ◽  
Wolfgang Mühlbacher ◽  
Patrick Cramer
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Catalytic Mechanism ◽  
Protein Complexes ◽  
Hydrophobic Pocket ◽  
Pol Ii ◽  
Content Type ◽  
Open Conformation ◽  
Peptide Release ◽  
Hydrophobic Peptide

Biogenesis of the 12-subunit RNA polymerase II (Pol II) transcription complex requires so-called GPN-loop GTPases, but the function of these enzymes is unknown. Here we report the first crystal structure of a eukaryotic GPN-loop GTPase, theSaccharomyces cerevisiaeenzyme Npa3 (a homolog of human GPN1, also called RPAP4, XAB1, and MBDin), and analyze its catalytic mechanism. The enzyme was trapped in a GDP-bound closed conformation and in a novel GTP analog-bound open conformation displaying a conserved hydrophobic pocket distant from the active site. We show that Npa3 has chaperone activity and interacts with hydrophobic peptide regions of Pol II subunits that form interfaces in the assembled Pol II complex. Biochemical results are consistent with a model that the hydrophobic pocket binds peptides and that this can allosterically stimulate GTPase activity and subsequent peptide release. These results suggest that GPN-loop GTPases are assembly chaperones for Pol II and other protein complexes.

scholarly journals A slow kinetic transient in RNA synthesis catalysed by wheat-germ RNA polymerase II

1988 ◽  
Vol 253 (1) ◽  
pp. 281-285 ◽  
Author(s):  
C Job ◽  
L De Mercoyrol ◽  
D Job
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Conformational Change ◽  
Wheat Germ ◽  
Rna Synthesis ◽  
Single Step ◽  
Slow Kinetic ◽  
Productive Elongation

Progress curves of U-A-primed RNA synthesis catalysed by wheat-germ RNA polymerase II on a poly[d(A-T)] template exhibit a slow burst of activity. In contrast, the progress curves of single-step addition of UMP to U-A primer in the abortive elongation reaction do not exhibit the slow burst of activity. The correlation between the kinetic transient in the productive pathway of RNA synthesis and the rate of abortive elongation is suggestive of the occurrence of a slow conformational change of the transcription complex during the transition from abortive to productive elongation. The exceptional duration of the transient burst (in the region of 4 min) may suggest a transition of a hysteretic type.

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