Short communicationThe activator/repressor protein DnrO of Streptomyces peucetius binds to DNA without changing its topology
Introduction
Transcription initiation mainly depends on the promoter strength that determines the affinity of RNA polymerase to DNA. It has been shown that intrinsic curvature of the promoter sequence promotes transcription initiation [1]. Intrinsic bends act as docking site for RNA polymerase and transcription factors [2]. Besides intrinsic bending, activator proteins are also known to induce DNA bending in and around promoter region [3]. Protein-induced bending facilitates stable binding of RNA polymerase to promoter [4]. Though not examined in all transcription factors, it seems that most of them bend DNA to different extent [5].
Daunorubicin (DNR) is an antitumour chemotherapeutic agent synthesized by a type II polyketide pathway of Streptomyces peucetius. Regulation of this pathway is by three genes dnrO [6], dnrN [7] and dnrI [8]. DnrO and dnrN share a common upstream intergenic DNA sequence and are divergently transcribed as shown in Fig. 1. The first gene to be activated in the pathway is dnrO by unknown means. DnrO apparently binds to its cognate sequence (37 bp sequence that overlaps with dnrO's P1 promoter) within the 433 bp intergenic region between dnrN and dnrO genes to activate dnrN [9]. Binding of DnrO to its cognate sequence (promoter/operator) is inhibited by DNR [9]. Feedback inhibition of DNR biosynthesis has been demonstrated in S. peucetius by the formation of drug–protein complex between DNR and DnrO [9]. DNR–DnrO complex fails to bind at its sequence and thereby dnrN is not activated. DnrN is a pseudo response regulatory protein that binds upstream to the un-translated region of dnrI gene – the key regulator – and activates it [7]. DnrI binds to several upstream elements of structural genes for the activation DNR biosynthesis [8]. The dnrN–dnrO intergenic region comprises of three active promoters for dnrO and one promoter for dnrN as shown in Fig. 1. The 5′ un-translated region of dnrN overlaps with the transcripts from OP2 and OP3 (promoters P2 and P3 of dnrO) promoters of dnrO [6].
DnrO protein belongs to DeoR (deo operon repressor) family of transcriptional regulators and possesses HTH DNA binding domain at the N-terminal end [6]. Though DeoR family of proteins are usually transcriptional repressors, DnrO seem to be exceptional as it performs activation and repression. The biotin operon repressor BirA of E. coli has a DNA binding domain structurally similar to DnrO. However, BirA binds to DNA as a dimer unlike DnrO that binds as monomer [10]. DnrO attracts special interest due to its dual role as activator and repressor; therefore understanding its DNA binding property will provide insight into the functional role of this protein. Homolog of DnrO has not been found so far either in Streptomyces or in any other organism, suggesting that it could be a novel transcriptional regulator.
Circular permutation assay detects DNA deformation by differential electrophoretic mobility of protein–DNA complexes. DNA fragments with a protein-induced distortion in the middle of the molecule have a different shape, and hence different electrophoretic mobility, compared to DNA fragments of identical length and composition with a distortion near one end [3]. The detailed relationship between electrophoretic mobility and conformation is complex; however, there are a few simple algorithms that allow to map the locus of protein–DNA interaction and to estimate the amount of distortion introduced in DNA [11]. BirA is known to introduce a bend of 40° at the target DNA [10]. This property of BirA prompted us to investigate the topological changes of DNA during interaction of DnrO. Using circular permutation gel shift assay, we found that DnrO does not bend the promoter/operator sequence and the activation of dnrN must be DNA topology independent.
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Enzymes and chemicals
Restriction enzymes (Promega, Madison, USA), Taq polymerase (Invitrogen, Paisley, UK), T4 polynucleotide kinase (Genei, Bangalore, India) were used in this study. Fine chemicals and Ni-Agarose were purchased from Sigma Aldrich Chemicals Pvt. Ltd., India. Components of culture media were purchased from HiMedia Laboratories Pvt. Ltd., Mumbai, India. Gamma 33P ATP was obtained from Board of Radiation and Isotope Technology, Jonaki, Hyderabad, India. Other reagents were obtained from standard
Results and discussion
The dnrO gene was amplified using pdnrNO plasmid as template and cloned in pQE31 expression vector. The recombinant plasmid was introduced into E. coli M 15 pREP4 cells. IPTG-induced E. coli culture was lysed and total protein profile in SDS-PAGE showed abundant expression of rDnrO. The solubility of the expressed protein was checked by centrifuging the intracellular proteins at 1000 rpm for 10 min at 4 °C. The supernatant and the pellet fractions were checked by SDS-PAGE. Predominant portion of
Acknowledgements
Authors thank Department of Biotechnology, Government of India for financial support. Additional funds from UPE project of Madurai Kamaraj University (MKU), India supported by University Grants Commission, India is acknowledged. Authors thank Prof. K. Dharmalingam for his critical comments and technical support. Instrument support given by DBT Centre for Genetic Engineering and Strain Manipulation, at MKU is acknowledged.
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