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 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 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.

scholarly journals Complex RNA chain elongation kinetics by wheat germ RNA polymerase II

1984 ◽  
Vol 12 (7) ◽  
pp. 3303-3320 ◽  
Author(s):  
Dominique Job ◽  
Robert Durand ◽  
Claudette Job ◽  
Marcel Teissere
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Wheat Germ ◽  
Chain Elongation

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 Kinetic co-operativity of wheat-germ RNA polymerase II with adenosine 5′-[βγ-imido]triphosphate as substrate

1988 ◽  
Vol 252 (1) ◽  
pp. 55-63 ◽  
Author(s):  
C Job ◽  
J M Soulié ◽  
D Job
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Kinetic Models ◽  
Metal Ion ◽  
Wheat Germ ◽  
Substrate Recognition ◽  
Chain Elongation ◽  
Velocity Versus ◽  
Natural Substrate ◽  
Alternative Substrate

A kinetic study of productive RNA chain elongation indicates that adenosine 5′-[beta gamma-imido]triphosphate can serve as substrate in reactions catalysed by purified wheat-germ RNA polymerase II on a poly[d(A-T)] template. However, in contrast with the results obtained with the natural substrate ATP, the double-reciprocal plots, 1/velocity versus 1/[nucleotide], are not linear but characteristic of apparent negative co-operativity. The extent of the kinetic co-operativity is modified when the reactions are conducted in the additional presence of fixed amounts of an alternative substrate such as ATP or inhibitors such as dATP or cordycepin triphosphate. Analogous results are obtained whether the reactions are carried out in the presence of Mg2+ or Mn2+ as the metal ion cofactor. However, the data show that with Mn2+ the RNA polymerase is less specific in substrate recognition than with Mg2+. Tentative kinetic models are proposed to account for the rate measurements.

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.

Mapping mutations in genes encoding the two large subunits of Drosophila RNA polymerase II defines domains essential for basic transcription functions and for proper expression of developmental genes

1993 ◽  
Vol 13 (7) ◽  
pp. 4214-4222
Author(s):  
Y Chen ◽  
J Weeks ◽  
M A Mortin ◽  
A L Greenleaf
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Dna Sequence ◽  
Pcr Amplification ◽  
Single Strand Conformation Polymorphism ◽  
Chain Elongation ◽  
Developmental Defect ◽  
Genes Encoding ◽  
Transcript Elongation

We have mapped a number of mutations at the DNA sequence level in genes encoding the largest (RpII215) and second-largest (RpII140) subunits of Drosophila melanogaster RNA polymerase II. Using polymerase chain reaction (PCR) amplification and single-strand conformation polymorphism (SSCP) analysis, we detected 12 mutations from 14 mutant alleles (86%) as mobility shifts in nondenaturing gel electrophoresis, thus localizing the mutations to the corresponding PCR fragments of about 350 bp. We then determined the mutations at the DNA sequence level by directly subcloning the PCR fragments and sequencing them. The five mapped RpII140 mutations clustered in a C-terminal portion of the second-largest subunit, indicating the functional importance of this region of the subunit. The RpII215 mutations were distributed more broadly, although six of eight clustered in a central region of the subunit. One notable mutation that we localized to this region was the alpha-amanitin-resistant mutation RpII215C4, which also affects RNA chain elongation in vitro. RpII215C4 mapped to a position near the sites of corresponding mutations in mouse and in Caenorhabditis elegans genes, reinforcing the idea that this region is involved in amatoxin binding and transcript elongation. We also mapped mutations in both RpII215 and RpII140 that cause a developmental defect known as the Ubx effect. The clustering of these mutations in each gene suggests that they define functional domains in each subunit whose alteration induces the mutant phenotype.

Sign in / Sign up

Export Citation Format

Share Document