IN VITRO TRANSCRIPTION OF Ad2 DNA BY EUKARYOTIC RNA POLYMERASES II. I. KINETICS OF FORMATION OF STABLE BINARY COMPLEXES AT SPECIFIC SITES

The recent success in reconstitution of RNAPs (RNA polymerases) from hyperthermophilic archaea from bacterially expressed purified subunits opens the way for detailed structure–function analyses of multisubunit RNAPs. The archaeal enzyme shows close structural similarity to eukaryotic RNAP, particularly to polymerase II, and can therefore be used as model for analyses of the eukaryotic transcriptional machinery. The cleft loops in the active centre of RNAP were deleted and modified to unravel their function in interaction with nucleic acids during transcription. The rudder, lid and fork 2 cleft loops were required for promoter-directed initiation and elongation, the rudder was essential for open complex formation. Analyses of transcripts from heteroduplex templates containing stable open complexes revealed that bubble reclosure is required for RNA displacement during elongation. Archaeal transcription systems contain, besides the orthologues of the eukaryotic transcription factors TBP (TATA-box-binding protein) and TF (transcription factor) IIB, an orthologue of the N-terminal part of the α subunit of eukaryotic TFIIE, called TFE, whose function is poorly understood. Recent analyses revealed that TFE is involved in open complex formation and, in striking contrast with eukaryotic TFIIE, is also present in elongation complexes. Recombinant archaeal RNAPs lacking specific subunits were used to investigate the functions of smaller subunits. These studies revealed that the subunits P and H, the orthologues of eukaryotic Rpb12 and Rpb5, were not required for RNAP assembly. Subunit P was essential for open complex formation, and the ΔH enzyme was greatly impaired in all assays, with the exception of promoter recruitment. Recent reconstitution studies indicate that Rpb12 and Rpb5 can be incorporated into archaeal RNAP and can complement for the function of the corresponding archaeal subunit in in vitro transcription assays.

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Hybrid Bordetella pertussis-Escherichia coli RNA Polymerases: Selectivity of Promoter Activation

Journal of Bacteriology ◽

10.1128/jb.180.6.1567-1569.1998 ◽

1998 ◽

Vol 180 (6) ◽

pp. 1567-1569 ◽

Cited By ~ 9

Author(s):

Pierre Steffen ◽

Agnes Ullmann

Keyword(s):

Escherichia Coli ◽

Bordetella Pertussis ◽

In Vitro Transcription ◽

Rna Polymerases ◽

Α Subunit ◽

E Coli ◽

Transcription System ◽

Catabolite Gene Activator Protein ◽

Transcription Activators

ABSTRACT We constructed hybrid Bordetella pertussis-Escherichia coli RNA polymerases and compared productive interactions between transcription activators and cognate RNA polymerase subunits in an in vitro transcription system. Virulence-associated genes of B. pertussis, in the presence of their activator BvgA, are transcribed by all variants of hybrid RNA polymerases, whereas transcription at the E. coli lacpromoter regulated by the cyclic AMP-catabolite gene activator protein has an absolute requirement for the E. coli α subunit. This suggests that activator contact sites involve a high degree of selectivity.

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Mutational Analysis of the Chlamydia trachomatis rRNA P1 Promoter Defines Four Regions Important for Transcription In Vitro

Journal of Bacteriology ◽

10.1128/jb.180.9.2359-2366.1998 ◽

1998 ◽

Vol 180 (9) ◽

pp. 2359-2366 ◽

Cited By ~ 26

Author(s):

Ming Tan ◽

Tamas Gaal ◽

Richard L. Gourse ◽

Joanne N. Engel

Keyword(s):

Escherichia Coli ◽

Chlamydia Trachomatis ◽

Rna Polymerase ◽

Mutational Analysis ◽

In Vitro Transcription ◽

Rna Polymerases ◽

Rich Region ◽

E Coli ◽

Transcription In Vitro

ABSTRACT We have characterized the Chlamydia trachomatisribosomal promoter, rRNA P1, by measuring the effect of substitutions and deletions on in vitro transcription with partially purifiedC. trachomatis RNA polymerase. Our analyses indicate that rRNA P1 contains potential −10 and −35 elements, analogous toEscherichia coli promoters recognized by E-ς70. We identified a novel AT-rich region immediately downstream of the −35 region. The effect of this region was specific for C. trachomatis RNA polymerase and strongly attenuated by single G or C substitutions. Upstream of the −35 region was an AT-rich sequence that enhanced transcription by C. trachomatis and E. coli RNA polymerases. We propose that this region functions as an UP element.

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Effect of estrogen on gene expression in the chick oviduct. Kinetics of initiation of in vitro transcription on chromatin.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(17)33811-5 ◽

1976 ◽

Vol 251 (4) ◽

pp. 1137-1146

Author(s):

M Hirose ◽

M J Tsai ◽

B W O'Malley

Keyword(s):

Gene Expression ◽

In Vitro Transcription ◽

Kinetics Of

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Discrimination between G/C Binding Sites by Olivomycin A Is Determined by Kinetics of the Drug-DNA Interaction

International Journal of Molecular Sciences ◽

10.3390/ijms21155299 ◽

2020 ◽

Vol 21 (15) ◽

pp. 5299

Author(s):

Artemy D. Beniaminov ◽

Galina V. Chashchina ◽

Mikhail A. Livshits ◽

Olga I. Kechko ◽

Vladimir A. Mitkevich ◽

...

Keyword(s):

Binding Sites ◽

Dna Interaction ◽

In Vitro Transcription ◽

Dissociation Rate ◽

Titration Calorimetry ◽

Drug Candidates ◽

Important Approach ◽

Functional Relevance ◽

Kinetics Of

Olivomycin A (OA) exerts its cytotoxic potency due to binding to the minor groove of the G/C-rich DNA and interfering with replication and transcription. Screening of the complete set of tetranucleotide G/C sites by electrophoretic mobility gel shift assay (EMSA) revealed that the sites containing central GC or GG dinucleotides were able to bind OA, whereas the sites with the central CG dinucleotide were not. However, studies of equilibrium OA binding in solution by fluorescence, circular dichroism and isothermal titration calorimetry failed to confirm the sequence preference of OA, indicating instead a similar type of complex and comparable affinity of OA to all G/C binding sites. This discrepancy was resolved by kinetics analysis of the drug–DNA interaction: the dissociation rate significantly differed between SGCS, SGGS and SCGS sites (S stands for G or C), thereby explaining the disintegration of the complexes during EMSA. The functional relevance of the revealed differential kinetics of OA–DNA interaction was demonstrated in an in vitro transcription assay. These findings emphasize the crucial role of kinetics in the mechanism of OA action and provide an important approach to the screening of new drug candidates.

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Defining the divergent enzymatic properties of RNA polymerases I and II

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.015904 ◽

2020 ◽

pp. jbc.RA120.015904

Author(s):

Ruth Q. Jacobs ◽

Zachariah M Ingram ◽

Aaron L. Lucius ◽

David A. Schneider

Keyword(s):

Nuclear Dna ◽

In Vitro Transcription ◽

Rna Polymerases ◽

Enzymatic Properties ◽

Experimental Conditions ◽

Pol Ii ◽

Pol I ◽

Nucleotide Addition ◽

Addition Rate

Eukaryotes express at least three nuclear DNA-dependent RNA polymerases (Pols) responsible for synthesizing all RNA required by the cell. Despite sharing structural homology, they have functionally diverged to suit their distinct cellular roles. Although the Pols have been studied extensively, direct comparison of their enzymatic properties is difficult since studies are often conducted under disparate experimental conditions and techniques. Here, we directly compare and reveal functional differences between Saccharomyces cerevisiae Pols I and II using a series of quantitative in vitro transcription assays. We find that Pol I single and multi-nucleotide addition rate constants are faster than those of Pol II. Pol I elongation complexes (ECs) are less stable than Pol II ECs, and Pol I is more error prone than Pol II. Collectively, these data show that the enzymatic properties of the Pols have diverged over the course of evolution, optimizing these enzymes for their unique cellular responsibilities.

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