Purification and DNA-Binding Properties of RNA Polymerase from Bacillus subtilis

ABSTRACT The N-terminal region (region 1.1) of σ70, the primary σ subunit of Escherichia coli RNA polymerase, is a negatively charged domain that affects the DNA binding properties of σ70 regions 2 and 4. Region 1.1 prevents the interaction of free σ70 with DNA and modulates the formation of stable (open) polymerase/promoter complexes at certain promoters. The bacteriophage T4 AsiA protein is an inhibitor of σ70-dependent transcription from promoters that require an interaction between σ70 region 4 and the −35 DNA element and is the coactivator of transcription at T4 MotA-dependent promoters. Like AsiA, the T4 activator MotA also interacts with σ70 region 4. We have investigated the effect of region 1.1 on AsiA inhibition and MotA/AsiA activation. We show that σ70 region 1.1 is not required for MotA/AsiA activation at the T4 middle promoter P uvsX . However, the rate of AsiA inhibition and of MotA/AsiA activation of polymerase is significantly increased when region 1.1 is missing. We also find that RNA polymerase reconstituted with σ70 that lacks region 1.1 is less stable than polymerase with full-length σ70. Our previous work has demonstrated that the AsiA-inhibited polymerase is formed when AsiA binds to region 4 of free σ70 and then the AsiA/σ70 complex binds to core. Our results suggest that in the absence of region 1.1, there is a shift in the dynamic equilibrium between polymerase holoenzyme and free σ70 plus core, yielding more free σ70 at any given time. Thus, the rate of AsiA inhibition and AsiA/MotA activation increases when RNA polymerase lacks region 1.1 because of the increased availability of free σ70. Previous work has argued both for and against a direct interaction between regions 1.1 and 4. Using an E. coli two-hybrid assay, we do not detect an interaction between these regions. This result supports the idea that the ability of region 1.1 to prevent DNA binding by free σ70 arises through an indirect effect.

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The −10 Region Is a Key Promoter Specificity Determinant for the Bacillus subtilis Extracytoplasmic-Function ς Factors ςX and ςW

Journal of Bacteriology ◽

10.1128/jb.183.6.1921-1927.2001 ◽

2001 ◽

Vol 183 (6) ◽

pp. 1921-1927 ◽

Cited By ~ 49

Author(s):

Jian Qiu ◽

John D. Helmann

Keyword(s):

Bacillus Subtilis ◽

Amino Acid ◽

Rna Polymerase ◽

Context Effects ◽

Promoter Strength ◽

Binding Properties ◽

Promoter Specificity ◽

Dna Binding Properties

ABSTRACT Transcriptional selectivity derives, in large part, from the sequence-specific DNA-binding properties of the ς subunit of RNA polymerase. There are 17 ς factors in Bacillus subtilis which, in general, recognize distinct sets of promoters. However, some ς factors have overlapping promoter selectivity. We hypothesize that the overlap between the regulons activated by the ςX and ςW factors can be explained by overlapping specificity for the −10 region: ςX recognizes −10 elements with the sequence CGAC and ςW recognizes CGTA, while both can potentially recognize CGTC. To test this model, we mutated the ςX-specific autoregulatory site (PX), containing the −10 element CGAC, to either CGTC or GCTA. Conversely, the ςW autoregulatory site (PW) was altered from CGTA to CGTC or CGAC. Transcriptional analyses, both in vitro and in vivo, indicate that changes to the −10 element are sufficient to switch a promoter from the ςX to the ςW regulon or, conversely, from the ςW to the ςX regulon, but context effects clearly play an important role in determining promoter strength. It seems likely that these subtle differences in promoter selectivity derive from amino acid differences in conserved region 2 of ς, which contacts the −10 element. However, we were unable to alter promoter selectivity by replacements of two candidate recognition residues in ςW.

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DNA-Binding Properties of the Bacillus subtilis and Aeribacillus pallidus AC6 D Proteins

Journal of Bacteriology ◽

10.1128/jb.01193-10 ◽

2010 ◽

Vol 193 (2) ◽

pp. 575-579 ◽

Cited By ~ 7

Author(s):

E. Sevim ◽

A. Gaballa ◽

A. O. Belduz ◽

J. D. Helmann

Keyword(s):

Bacillus Subtilis ◽

Dna Binding ◽

Binding Properties ◽

Dna Binding Properties

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Yeast RNA polymerase III. Chromatographic, catalytic and DNA-binding properties are highly dependent on the type of anion

European Journal of Biochemistry ◽

10.1111/j.1432-1033.1984.tb08198.x ◽

1984 ◽

Vol 141 (2) ◽

pp. 345-350 ◽

Cited By ~ 3

Author(s):

Odd Stokke GABRIELSEN ◽

Kjersti Elisabeth ANDERSEN ◽

Tordis Beate OYEN

Keyword(s):

Dna Binding ◽

Rna Polymerase ◽

Rna Polymerase Iii ◽

Binding Properties ◽

Polymerase Iii ◽

Dna Binding Properties

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Noncanonical DNA-binding properties of potential components of an antitumor composition (cytokine AIMP1/p43, lectin SNA, izatizon), which are able to affect different targets

Faktori eksperimental'noi evolucii organizmiv ◽

10.7124/feeo.v22.969 ◽

2018 ◽

Vol 22 ◽

pp. 323-328

Author(s):

O. Yu. Tsuvariev ◽

O. V. Starozhuk ◽

I. S. Karpova ◽

L. G. Palchykovska ◽

L. A. Zayika ◽

...

Keyword(s):

Dna Binding ◽

Rna Polymerase ◽

Plasmid Dna ◽

Structural Changes ◽

In Vitro Transcription ◽

Test System ◽

Binding Properties ◽

Experimental Conditions ◽

Dna Binding Properties

Aim. To search the DNA-binding properties of various drugs promising for the creation of the antitumor composition (AIMP1/p43, SNA-I, izatizon), as well as their ability to influence on key matrix processes – DNA transcription and replication, which are a sensitive target for the action of many pharmacological drugs. Methods. The recombinant protein AIMP1/p43 was obtained from supernatant of E.coli lysed cells by metallic chelating chromatography on a Ni‑NTA‑agarose column. Also there were used the elderberry bark lectin SNA-I (LECTINOTEST, Ukraine), and the preparation of own production izatizon (IMBG NAS of Ukraine). The DNA-binding ability of the drugs was investigated by EMSA. As a transcription test system, an enzyme complex of DNA-dependent RNA polymerase of T7 bacteriophage and plasmid DNA pTZ19R * were used. PCR with Taq DNA polymerase and the plasmid pTZ19R * was used as the model replicative system. Results. Experimental conditions, in which the specificity of each drug action appeared, were found: the cytokine AIMP1/p43 and the SNA-I demonstrated the ability to bind to DNA by the EMSA test; izatizon induced structural changes in DNA, blocked the amplification of DNA during the PCR process, and also in vitro transcription with the participation of DNA-dependent RNA polymerase of the bacteriophage T7. Conclusions. Each of the selected drugs, which according to the literature data have antitumor potential, has demonstrated the ability to interact with the model supercoiled plasmid DNA pTZ19R *. The different ways of the selected drugs influence on DNA allows us to expect their effective combined action and synergism in case of their application as a common antitumor composition. Keywords: cytokine AIMP1/p43, SNA-I, izatizon, EMSA method, transcription inhibiting, amplification blocking.

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