Interaction of bacterial RNA-polymerase with two different promoters of phage T7 DNA. Conformational analysis

Ozoline, Olga N.; Uteshev, T.A.; Masulis, I. S.; Kamzolova, S. G.

doi:10.1016/0167-4781(93)90211-u

Cited by 17 publications

(18 citation statements)

References 27 publications

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“…DNAs fragments X1, X2 and X3 possess hexanucleotides with different degrees of homology to the canonical −10 and −35 elements of the σ 70 ‐RNAP of E. coli promoters. In addition X1 has, in the −44 region and downstream of the −10 element, sequences coincident with motifs pointed out by Ozoline et al [28,29] as frequently present in these E. coli promoters. The X8 fragment is particularly interesting because within it lies a putative promoter, which possesses most of the motifs described by Ozoline et al [28] as follows:…”

Section: Resultssupporting

confidence: 68%

A plasmid vector for isolation of strong promoters inEscherichia coli

Carbonelli¹,

Corley²,

Seigelchifer³

et al. 1999

FEMS Microbiology Letters

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In order to isolate very strong promoters from bacteria and bacteriophage a plasmid named pProm was constructed. It possesses an origin (ORI) for replication in Gram-negative bacteria, an ORI for replication in Gram-positive bacteria, a promoterless ampicillin resistance gene with a multiple cloning site (MCS) in the position formerly occupied by the ampicillin promoter, a tetracycline resistance gene for selection in Gram-negative bacteria and a chloramphenicol resistance gene for selection in Gram-positive bacteria. Insertion in the MCS of DNA fragments of Staphylococcus aureus bacteriophages resulted in isolation of several clones very resistant to ampicillin. The DNA fragments inserted in these recombinant plasmids were sequenced and all of them contained putative promoter motifs. Direct measurement of the penicillinase activity indicated that one of the isolated promoters could be included within a group of the stronger known prokaryotic promoters. According to these results pProm is a powerful tool to perform studies on promoter strength and for industrial applications.

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Section: Resultssupporting

confidence: 68%

A plasmid vector for isolation of strong promoters inEscherichia coli

Carbonelli¹,

Corley²,

Seigelchifer³

et al. 1999

FEMS Microbiology Letters

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“…Within the engineered integration plasmid pS‐T7A1‐chiR, the chiR gene was linked to the sequence of the E. coli phage T7A1 promoter, which has recently been shown to be active in myxobacteria (Julien, 2003; Julien and Fehd, 2003). T7A1 has been characterized as a constitutive promoter in other bacteria, because RNAP can initiate transcription in the absence of activators (Ozoline et al ., 1993). After integration of the plasmid into the chromosome, a merodiploid mutant carrying two functional copies of chiR was generated, in which the first copy of the gene was driven by the wild‐type promoter, whereas the second copy was dependent on T7A1.…”

Section: Discussionmentioning

confidence: 99%

Deciphering regulatory mechanisms for secondary metabolite production in the myxobacterium Sorangium cellulosum So ce56

Rachid

Gerth

Kochems

et al. 2007

Molecular Microbiology

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SummarySorangium cellulosum strains produce approximately 50% of the biologically active secondary metabolites known from myxobacteria. These metabolites include several compounds of biotechnological importance such as the epothilones and chivosazols, which, respectively, stabilize the tubulin and actin skeletons of eukaryotic cells. S. cellulosum is characterized by its slow growth rate, and natural products are typically produced in low yield. In this study, biomagnetic bead separation of promoter-binding proteins and subsequent inactivation experiments were employed to identify the chivosazol regulator, ChiR, as a positive regulator of chivosazol biosynthesis in the genome-sequenced strain So ce56. Overexpression of chiR under the control of T7A1 promoter in a merodiploid mutant resulted in fivefold overproduction of chivosazol in a kinetic shake flask experiment, and 2.5-fold overproduction by fermentation. Using quantitative reverse transcription PCR and gel shift experiments employing heterologously expressed ChiR, we have shown that transcription of the chivosazol biosynthetic genes (chiA-chiF) is directly controlled by this protein. In addition, we have demonstrated that ChiR serves as a pleiotropic regulator in S. cellulosum, because mutant strains lack the ability to develop into regular fruiting bodies.

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“…Modification of Mutant ␣-Subunits with FMA-The synthesis and spectral and chemical characteristics of FMA are described elsewhere (17,24,25). Since a mercaptide bond is unstable in the presence of thiol compounds, DTT was removed from all protein samples by gel filtration through a Sephadex G-50 column (1 ϫ 8 cm).…”

Section: Methodsmentioning

confidence: 99%

Transcription Activation Mediated by the Carboxyl-terminal Domain of the RNA Polymerase α-Subunit

Ozoline¹,

Fujita²,

Ishihama³

2000

Journal of Biological Chemistry

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Conformational changes within the carboxyl-terminal domain of theIn transcription initiation by the prokaryotic RNA polymerase, exchangeable -subunits are responsible for recognition of the core promoter DNA (1-3), whereas the carboxyl-terminal domain of the ␣-subunit (␣-CTD) 1 makes additional contacts with more distal regions (4). By both biochemical (4, 5) and physical (6, 7) data, the upstream sequence of the rrnBP1 promoter, generally designated as the UP element, was proven to directly contact ␣-CTD. ␣-CTD is also responsible for interaction with a set of transcription factors, designated as class I (or ␣-contact) factors, regulating transcription efficiency (3, 8). The most studied factor is cAMP receptor protein (CRP) (9 -11), which regulates transcription of Ͼ80 genes (12, 13). The molecular mechanism of transcription regulation by CRP depends on the position of its binding site on the promoter sequence. Promoters that have the CRP-binding site centered between positions Ϫ60 and Ϫ100 usually require ␣-CTD for activation (3,8). Random and site-directed mutagenesis within ␣-CTD revealed that the amino acid residues responsible for interaction with the UP element in rrnBP1 include Leu-260, Leu-262, Arg-265, Asn-268, Cys-269, and Lys-297, whereas Leu-260, Leu-262, Arg-265, Asn-268, Leu-270, Ile-275, Lys-297, and Lys-298 are involved in transcription activation at the lacP1 promoter by CRP (14 -16). The amino acid residues mostly important for UP element-dependent transcription (Leu-262, Arg-265, Asn-268, and Lys-297) are also crucial for CRP-dependent activation. Two possibilities are considered to explain this overlapping: (i) the same surface of ␣-CTD participates in the specific interaction with both the DNA UP element and CRP, or (ii) CRP ensures the contact between the specific surface of ␣-CTD and promoter DNA. These two possibilities are not necessarily contradictory if one ␣-subunit is involved in interaction with CRP while another forms a specific contact with DNA, or if ␣-CTD participates in an initial and transient interaction with CRP prior to the final and stable interaction with DNA.To get insight into the detailed mechanism of ␣-CTD interactions with the DNA UP element and CRP, we tried in this study to monitor CRP-induced conformational alterations in ␣-CTD upon transition from RNA polymerase-promoter binary complexes to ternary complexes. For this purpose, a set of single Cys mutant ␣-subunits at residues 263, 271, 272, 283, 285, 292, and 309 was constructed in addition to the Cys-269 mutant that was used in our previous studies (17,18), and a fluorescent probe, fluorescein mercuric acetate (FMA), was conjugated to each of the single Cys mutant ␣-subunits. Using the reconstituted RNA polymerase holoenzymes containing the FMA-tethered mutant ␣-subunits, the fluorescent spectral changes were monitored after complex formation with the CRPdependent promoter uxuAB, the factor-independent promoter T7D, and the UP element-dependent promoter rrnBP1. The topology of DNA contact surfaces was furthe...

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Interaction of bacterial RNA-polymerase with two different promoters of phage T7 DNA. Conformational analysis

Cited by 17 publications

References 27 publications

A plasmid vector for isolation of strong promoters inEscherichia coli

A plasmid vector for isolation of strong promoters inEscherichia coli

Deciphering regulatory mechanisms for secondary metabolite production in the myxobacterium Sorangium cellulosum So ce56

Transcription Activation Mediated by the Carboxyl-terminal Domain of the RNA Polymerase α-Subunit

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