scholarly journals Effect of Sarkosyl and heparin on single-step addition reactions catalysed by wheat-germ RNA polymerase II–poly[d(A–T)]transcription complexes

1989 ◽  
Vol 260 (3) ◽  
pp. 795-801 ◽  
Author(s):  
L De Mercoyrol ◽  
C Job ◽  
D Job
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Wheat Germ ◽  
Single Step ◽  
Addition Reactions ◽  
Phosphodiester Bond ◽  
Sizeable Fraction ◽  
Transcription Complexes ◽  
Enzyme Molecules ◽  
Dna Template

Incubation of purified wheat-germ RNA polymerase II with poly[d(A-T)] template, Mn2+, U-A dinucleoside monophosphate primer and UTP substrate resulted in catalytic formation of the trinucleoside diphosphate U-A-U, in accordance with the results of previous studies. Both Sarkosyl and heparin inhibited completely and immediately (within less than 1 min) U-A-U synthesis, if either of these compounds was added to the assays during the progress of the reaction. This behaviour is in marked contrast to that reported for single-step addition reactions catalysed by Escherichia coli RNA polymerase on the same template [Sylvester & Cashel (1980) Biochemistry 19, 1069-1074]. However, treatment of the transcription complexes with Sarkosyl or heparin for periods sufficient to abolish U-A-U formation completely did not suppress completely the ability of such complexes to elongate RNA chains. Hence, the effect of Sarkosyl or heparin on the rate of U-A-U synthesis was predominantly due to change in the rate (or in the mechanism) of trinucleotide product release by the transcription complexes. Furthermore, once U-A-U synthesis has begun on the poly[d(A-T)] template, the transcription complexes became resistant to the action of a competitor DNA such as poly[d(G-C)]. The results are consistent with a model where at least a sizeable fraction of the enzyme molecules remains associated with the DNA template upon formation of a single phosphodiester bond.

Transcription of left-handed Z-DNA templates: increased rate of single-step addition reactions catalyzed by wheat germ RNA polymerase II

Biochemistry ◽  
1988 ◽  
Vol 27 (17) ◽  
pp. 6371-6378 ◽  
Author(s):  
Dominique Job ◽  
Philippe Marmillot ◽  
Claudette Job ◽  
Thomas M. Jovin
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Wheat Germ ◽  
Single Step ◽  
Addition Reactions ◽  
Dna Templates ◽  
Left Handed ◽  
Z Dna

scholarly journals Studies on the inhibition by α-amanitin of single-step addition reactions and productive RNA synthesis catalysed by wheat-germ RNA polymerase II

1989 ◽  
Vol 258 (1) ◽  
pp. 165-169 ◽  
Author(s):  
L de Mercoyrol ◽  
C Job ◽  
D Job
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Wheat Germ ◽  
Rna Synthesis ◽  
Single Step ◽  
Chain Elongation ◽  
Higher Plant ◽  
Experimental Conditions ◽  
Phosphodiester Bond ◽  
Alpha Amanitin

The rate of formation of a single phosphodiester bond with UTP substrate, U-A primer, poly[d(A-T)] template and wheat-germ RNA polymerase II is greatly depressed in the presence of alpha-amanitin. Half-maximal inhibition occurs at 0.04 microgram/ml, in close agreement with published values for inhibition of productive RNA synthesis with class II RNA polymerases from higher-plant species. However, a sizeable proportion of U-A-U synthesis is resistant to inhibition by excess alpha-amanitin. In the additional presence of ATP, i.e. under experimental conditions permitting RNA chain elongation, the synthesis of poly[r(A-U)] is arrested after the formation of the first phosphodiester bond. The results support the contention that the main enzymic process disrupted by alpha-amanitin is the translocation step of the transcription complex along the DNA template.

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.

scholarly journals Abortive intermediates in transcription by wheat-germ RNA polymerase II. Dynamic aspects of enzyme/template interactions in selection of the enzyme synthetic mode

1990 ◽  
Vol 269 (3) ◽  
pp. 651-658 ◽  
Author(s):  
L de Mercoyrol ◽  
J M Soulié ◽  
C Job ◽  
D Job ◽  
C Dussert ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Wheat Germ ◽  
Length Distribution ◽  
Enzyme Concentration ◽  
Single Step ◽  
Chain Elongation ◽  
Polymeric Product ◽  
Chain Length Distribution ◽  
Kinetics Of

At constant enzyme concentration and with the full set of nucleotide substrates dictated by template sequence, the chain-length distribution of polymeric product varies with template concentration in reactions catalysed by wheat-germ RNA polymerase II. Under the same conditions, but in the presence of a single ribonucleoside triphosphate, the rate of condensation of the triphosphate substrate to a dinucleotide primer also exhibits a complex dependence with the template concentration. This effect is observed using poly[d(A-T)] as a template. For both reactions there are two extreme types of behaviour in each of which transcription appears to involve a single enzyme synthetic mode, characterized by either a high (at low template concentration) or a low (at high template concentration) probability of releasing the transcripts. A strong correlation is found between these two pathways, such that conditions favouring the abortive release of trinucleotide products in the single-step addition reaction are associated with the synthesis of short-length RNA species in productive elongation, and reciprocally. A model previously developed by Papanicolaou, Lecomte & Ninio [(1986) J. Mol. Biol. 189, 435-448] to account for the kinetics of polymerization/excision ratios with Escherichia coli DNA polymerase I, and by Job, Soulié, Job & Shire [(1988) J. Theor. Biol. 134, 273-289] for kinetics of RNA-chain elongation by wheat-germ RNA polymerase II provides an explanation for the observed behaviour with the plant transcriptase. The basic requirement of this model is a slow equilibrium between two states of the polymerization complex with distinct probabilities of releasing the product. In the presence of Mn2+, and under conditions allowing the synthesis of poly[r(A-U)], one of these states is involved in the formation of oligonucleotides shorter than 15 bases, whereas the other catalyses the polymerization of chains longer than 40 bases.

scholarly journals Effect of low nucleotide concentrations on abortive elongation catalysed by wheat-germ RNA polymerase II

1987 ◽  
Vol 244 (1) ◽  
pp. 151-157 ◽  
Author(s):  
C Job ◽  
J Dietrich ◽  
D Shire ◽  
M Teissere ◽  
D Job
Keyword(s):  
Escherichia Coli ◽  
Substrate Specificity ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Dna Sequence ◽  
Wheat Germ ◽  
Starting Point ◽  
Dna Template ◽  
Plant Enzyme ◽  
Productive Elongation

A kinetic study of the effect of elongating nucleotide concentration on the reactions of abortive elongation catalysed by wheat-germ RNA polymerase II on a poly[d(A-T)] template suggests that the shift from abortive to productive elongation may involve the participation of at least two nucleotides, according to a mechanism very similar to that reported for Escherichia coli RNA polymerase. Experiments performed with non-complementary nucleotides with respect to the DNA template, and with substrate derivatives, allow an analysis of the substrate specificity during these reactions. Similar experiments performed with poly[d(A-A-T)].poly[d(T-T-A)] as template provide a starting point for a better understanding of the effect of DNA sequence on the rates of abortive and productive elongation catalysed by the plant enzyme.

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.

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