scholarly journals Quantitative analysis of the ternary complex of RNA polymerase, cyclic AMP receptor protein and DNA by fluorescence anisotropy measurements.

2008 ◽  
Vol 55 (3) ◽  
pp. 537-547
Author(s):  
Piotr Bonarek ◽  
Sylwia Kedracka-Krok ◽  
Barbara Kepys ◽  
Zygmunt Wasylewski
Keyword(s):  
Rna Polymerase ◽  
Fluorescence Anisotropy ◽  
Multicomponent System ◽  
Binding Constants ◽  
Receptor Protein ◽  
Activator Protein ◽  
Apparent Binding Constant ◽  
Transcription Complexes ◽  
Promoter Interaction

The in vitro formation of transcription complexes with Escherichia coli RNA polymerase was monitored using fluorescence anisotropy measurements of labeled fragments of DNA. The multicomponent system consisted of holo or core RNA polymerase (RNAP) and lac or gal promoter fragments of DNA (in different configurations), in the presence or absence of CRP activator protein (wt or mutants) with its ligand, cAMP. Values of the apparent binding constants characterizing the system were obtained, as a result of all processes taking place in the system. The interaction of the promoters with core RNAP in the absence of CRP protein was characterized by apparent binding constants of 0.67 and 1.9 x 10(6) M(-1) for lac166 and gal178, respectively, and could be regarded as nonspecific. The presence of wt CRP enhanced the strength of the interaction of core RNAP with the promoter, and even in the case of gal promoter it made this interaction specific (apparent binding constant 2.93 x 10(7) M(-1)). Holo RNAP bound the promoters significantly more strongly than core RNAP did (apparent binding constants 1.46 and 40.14 x 10(6) M(-1) for lac166 and gal178, respectively), and the presence of CRP also enhanced the strength of these interactions. The mutation in activator region 1 of CRP did not cause any significant disturbances in the holo RNAP-lac promoter interaction, but mutation in activator region 2 of the activator protein substantially weakened the RNAP-gal promoter interaction.

RNAP subunits F/E (RPB4/7) are stably associated with archaeal RNA polymerase: using fluorescence anisotropy to monitor RNAP assembly in vitro

2009 ◽  
Vol 421 (3) ◽  
pp. 339-343 ◽  
Author(s):  
Dina Grohmann ◽  
Angela Hirtreiter ◽  
Finn Werner
Keyword(s):  
Rna Polymerase ◽  
Molecular Interactions ◽  
Fluorescence Anisotropy ◽  
Rna Polymerases ◽  
Dynamic Exchange ◽  
Transcription Cycle

Archaeal and eukaryotic RNAPs (DNA-dependent RNA polymerases) are complex multi-subunit enzymes. Two of the subunits, F and E, which together form the F/E complex, have been hypothesized to associate with RNAP in a reversible manner during the transcription cycle. We have characterized the molecular interactions between the F/E complex and the RNAP core. F/E binds to RNAP with submicromolar affinity and is not in a dynamic exchange with unbound F/E.

scholarly journals Silk gland-specific tRNA(Ala) genes interact more weakly than constitutive tRNA(Ala) genes with silkworm TFIIIB and polymerase III fractions.

1994 ◽  
Vol 14 (3) ◽  
pp. 1806-1814 ◽  
Author(s):  
H S Sullivan ◽  
L S Young ◽  
C N White ◽  
K U Sprague
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Iii ◽  
Silk Gland ◽  
Flanking Sequence ◽  
Transcription Machinery ◽  
Polymerase Iii ◽  
Active Transcription ◽  
The Difference ◽  
Transcription Complexes

Constitutive and silk gland-specific tRNA(Ala) genes from silkworms have very different transcriptional properties in vitro. Typically, the constitutive type, which encodes tRNA(AlaC), directs transcription much more efficiently than does the silk gland-specific type, which encodes tRNA(AlaSG). We think that the inefficiency of the tRNA(AlaCG) gene underlies its capacity to be turned off in non-silk gland cells. An economical model is that the tRNA(AlaSG) promoter interacts poorly, relative to the tRNA(AlaC) promoter, with one or more components of the basal transcription machinery. As a consequence, the tRNA(AlaSG) gene directs the formation of fewer transcription complexes or of complexes with reduced cycling ability. Here we show that the difference in the number of active transcription complexes accounts for the difference in tRNA(AlaC) and tRNA(AlaSG) transcription rates. To determine whether a particular component of the silkworm transcription machinery is responsible for reduced complex formation on the tRNA(AlaSG) gene, we measured competition by templates for defined fractions of this machinery. We find that the tRNA(AlaSG) gene is greatly impaired, in comparison with the tRNA(AlaC) gene, in competition for either TFIIIB or RNA polymerase III. Competition for each of these fractions is also strongly influenced by the nature of the 5' flanking sequence, the promoter element responsible for the distinctive transcriptional properties of tRNA(AlaSG) and tRNA(AlaC) genes. These results suggest that differential interaction with TFIIIB or RNA polymerase III is a critical functional distinction between these genes.

scholarly journals Cyclic AMP Receptor Protein-Dependent Activation of the Escherichia coli acsP2 Promoter by a Synergistic Class III Mechanism

2003 ◽  
Vol 185 (17) ◽  
pp. 5148-5157 ◽  
Author(s):  
Christine M. Beatty ◽  
Douglas F. Browning ◽  
Stephen J. W. Busby ◽  
Alan J. Wolfe
Keyword(s):  
Escherichia Coli ◽  
Cyclic Amp ◽  
Rna Polymerase ◽  
Class I ◽  
Receptor Protein ◽  
Class Iii ◽  
Α Subunit ◽  
Cyclic Amp Receptor Protein ◽  
Rna Polymerase Holoenzyme

ABSTRACT The cyclic AMP receptor protein (CRP) activates transcription of the Escherichia coli acs gene, which encodes an acetate-scavenging enzyme required for fitness during periods of carbon starvation. Two promoters direct transcription of acs, the distal acsP1 and the proximal acsP2. In this study, we demonstrated that acsP2 can function as the major promoter and showed by in vitro studies that CRP facilitates transcription by “focusing” RNA polymerase to acsP2. We proposed that CRP activates transcription from acsP2 by a synergistic class III mechanism. Consistent with this proposal, we showed that CRP binds two sites, CRP I and CRP II. Induction of acs expression absolutely required CRP I, while optimal expression required both CRP I and CRP II. The locations of these DNA sites for CRP (centered at positions −69.5 and −122.5, respectively) suggest that CRP interacts with RNA polymerase through class I interactions. In support of this hypothesis, we demonstrated that acs transcription requires the surfaces of CRP and the C-terminal domain of the α subunit of RNA polymerase holoenzyme (α-CTD), which is known to participate in class I interactions: activating region 1 of CRP and the 287, 265, and 261 determinants of the α-CTD. Other surface-exposed residues in the α-CTD contributed to acs transcription, suggesting that the α-CTD may interact with at least one protein other than CRP.

scholarly journals RNA polymerase II elongation complexes paused after the synthesis of 15- or 35-base transcripts have different structures

1991 ◽  
Vol 11 (3) ◽  
pp. 1508-1522
Author(s):  
S C Linn ◽  
D S Luse
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Adenovirus Type ◽  
Dnase I ◽  
Transcription Complexes ◽  
Adenovirus Type 2

We have purified specific RNA polymerase II elongation intermediates initiated at the adenovirus type 2 major late promoter and paused either 15 or 35 to 36 bases downstream of the transcription initiation site. Transcription was arrested at these two sites by combining modification of the promoter sequence with limitation of appropriate nucleotide concentrations in the in vitro reaction. The resultant complexes were remarkably stable and could be purified away from free DNA and contaminating protein-DNA complexes, without loss of activity, by the use of sucrose gradient sedimentation and low-ionic-strength polyacrylamide gel electrophoresis. The complexes were characterized by both DNase I and o-phenanthroline-copper ion nuclease protection assays. The DNase I footprints revealed that the structures of the 15- and 35- to 36-nucleotide transcription complexes differed from those previously reported for an adenovirus type 2 major late preinitiation complex and a subsequent intermediate formed upon addition of ATP. Furthermore, the 35- to 36-nucleotide complex protected a significantly smaller portion of the template than the 15-nucleotide species and migrated at a slightly higher rate in polyacrylamide gels. These observations suggest that changes in structural organization may continue to occur in transcription complexes which are already committed to elongation.

Silk gland-specific tRNA(Ala) genes interact more weakly than constitutive tRNA(Ala) genes with silkworm TFIIIB and polymerase III fractions

1994 ◽  
Vol 14 (3) ◽  
pp. 1806-1814
Author(s):  
H S Sullivan ◽  
L S Young ◽  
C N White ◽  
K U Sprague
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Iii ◽  
Silk Gland ◽  
Flanking Sequence ◽  
Transcription Machinery ◽  
Polymerase Iii ◽  
Active Transcription ◽  
The Difference ◽  
Transcription Complexes

Constitutive and silk gland-specific tRNA(Ala) genes from silkworms have very different transcriptional properties in vitro. Typically, the constitutive type, which encodes tRNA(AlaC), directs transcription much more efficiently than does the silk gland-specific type, which encodes tRNA(AlaSG). We think that the inefficiency of the tRNA(AlaCG) gene underlies its capacity to be turned off in non-silk gland cells. An economical model is that the tRNA(AlaSG) promoter interacts poorly, relative to the tRNA(AlaC) promoter, with one or more components of the basal transcription machinery. As a consequence, the tRNA(AlaSG) gene directs the formation of fewer transcription complexes or of complexes with reduced cycling ability. Here we show that the difference in the number of active transcription complexes accounts for the difference in tRNA(AlaC) and tRNA(AlaSG) transcription rates. To determine whether a particular component of the silkworm transcription machinery is responsible for reduced complex formation on the tRNA(AlaSG) gene, we measured competition by templates for defined fractions of this machinery. We find that the tRNA(AlaSG) gene is greatly impaired, in comparison with the tRNA(AlaC) gene, in competition for either TFIIIB or RNA polymerase III. Competition for each of these fractions is also strongly influenced by the nature of the 5' flanking sequence, the promoter element responsible for the distinctive transcriptional properties of tRNA(AlaSG) and tRNA(AlaC) genes. These results suggest that differential interaction with TFIIIB or RNA polymerase III is a critical functional distinction between these genes.

scholarly journals Induction of Drosophila RNA polymerase III gene expression by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) is mediated by transcription factor IIIB.

1994 ◽  
Vol 14 (1) ◽  
pp. 339-347 ◽  
Author(s):  
M E Garber ◽  
A Vilalta ◽  
D L Johnson
Keyword(s):  
Rna Polymerase ◽  
Phorbol Ester ◽  
Rna Polymerase Iii ◽  
Protein Subunit ◽  
Cell Extracts ◽  
Polymerase Iii ◽  
Gene Complexes ◽  
Transcription Complexes ◽  
Dna Template

We have previously found that the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) induces specific transcription of tRNA and 5S RNA genes in Drosophila Schneider S-2 cells (M. Garber, S. Panchanathan, R. F. Fan, and D. L. Johnson, J. Biol. Chem. 266:20598-20601, 1991). Having derived cellular extracts from TPA-treated cells, that are capable of reproducing this stimulation in vitro, we have examined the mechanism for this regulatory event. Using conditions that limit reinitiation and produce single rounds of transcription from active gene complexes, we find that the number of functional transcription complexes is increased in extracts prepared from TPA-induced cells. We have analyzed the activities of the transcription factors TFIIIB and TFIIIC derived from extracts prepared from TPA-induced and noninduced cells. Examination of the relative activities of TFIIIC showed that both its ability to reconstitute transcription with TFIIIB and RNA polymerase III and its ability to stably bind to the DNA template are unchanged. However, the activity of TFIIIB derived from the TPA-induced cells is substantially increased compared with that derived from the noninduced cells. The differences in TFIIIB activity account for the differences in the overall transcriptional activities observed in the unfractionated extracts. Western blot analysis of the TATA-binding protein subunit of TFIIIB revealed that there is an increase in the amount of this polypeptide present in the induced cell extracts and TFIIIB fraction. Together, these results indicate that the TPA response in Drosophila cells stimulates specific transcription of RNA polymerase III genes by increasing the activity of the limiting transcription component, TFIIIB, and thereby increasing the number of functional transcription complexes.

scholarly journals Control of formation of two distinct classes of RNA polymerase II elongation complexes

1992 ◽  
Vol 12 (5) ◽  
pp. 2078-2090
Author(s):  
N F Marshall ◽  
D H Price
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Elongation Factor ◽  
In Vivo Experiments ◽  
Rapid Generation ◽  
Transcription Complexes ◽  
Productive Elongation

We have examined elongation by RNA polymerase II initiated at a promoter and have identified two classes of elongation complexes. Following initiation at a promoter, all polymerase molecules enter an abortive mode of elongation. Abortive elongation is characterized by the rapid generation of short transcripts due to pausing of the polymerase followed by termination of transcription. Termination of the early elongation complexes can be suppressed by the addition of 250 mM KCl or 1 mg of heparin per ml soon after initiation. Elongation complexes of the second class carry out productive elongation in which long transcripts can be synthesized. Productive elongation complexes are derived from early paused elongation complexes by the action of a factor which we call P-TEF (positive transcription elongation factor). P-TEF is inhibited by 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole at concentrations which have no effect on the initiation of transcription. By using templates immobilized on paramagnetic particles, we show that isolated preinitiation complexes lack P-TEF and give rise to transcription complexes which can carry out only abortive elongation. The ability to carry out productive elongation can be restored to isolated transcription complexes by the addition of P-TEF after initiation. A model is presented which describes the role of elongation factors in the formation and maintenance of elongation complexes. The model is consistent with the available in vivo data concerning control of elongation and is used to predict the outcome of other potential in vitro and in vivo experiments.

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