Twelve Species of the Nucleoid-associated Protein from Escherichia coli: SEQUENCE RECOGNITION SPECIFICITY AND DNA BINDING AFFINITY

Akira Ishihama,Talukder Ali Azam

doi:10.1074/jbc.274.46.33105

Akira Ishihama, Talukder Ali Azam

Open Access

https://doi.org/10.1074/jbc.274.46.33105

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Abstract

The genome of Escherichia coli is composed of a single molecule of circular DNA with the length of about 47,000 kilobase pairs, which is associated with about 10 major DNA-binding proteins, altogether forming the nucleoid. We expressed and purified 12 species of the DNA-binding protein, i.e. CbpA (curved DNA-binding protein A), CbpB or Rob (curved DNA-binding protein B or right arm of the replication origin binding protein), DnaA (DNA-binding protein A), Dps (DNA-binding protein from starved cells), Fis (factor for inversion stimulation), Hfq (host factor for phage Q(beta)), H-NS (histone-like nucleoid structuring protein), HU (heat-unstable nucleoid protein), IciA (inhibitor of chromosome initiation A), IHF (integration host factor), Lrp (leucine-responsive regulatory protein), and StpA (suppressor of td(-) phenotype A). The sequence specificity of DNA binding was determined for all the purified nucleoid proteins using gel-mobility shift assays. Five proteins (CbpB, DnaA, Fis, IHF, and Lrp) were found to bind to specific DNA sequences, while the remaining seven proteins (CbpA, Dps, Hfq, H-NS, HU, IciA, and StpA) showed apparently sequence-nonspecific DNA binding activities. Four proteins, CbpA, Hfq, H-NS, and IciA, showed the binding preference for the curved DNA. From the apparent dissociation constant (K(d)) determined using the sequence-specific or nonspecific DNA probes, the order of DNA binding affinity were determined to be: HU > IHF > Lrp > CbpB(Rob) > Fis > H-NS > StpA > CbpA > IciA > Hfq/Dps, ranging from 25 nM (HU binding to the non-curved DNA) to 250 nM (Hfq binding to the non-curved DNA), under the assay conditions employed.

Highlights

The genome of Escherichia coli is composed of a single molecule of circular DNA with the length of about 47,000 kilobase pairs, which is associated with about 10 major DNA-binding proteins, altogether forming the nucleoid
In order to understand the overall configuration and physiological activities of the E. coli genome under various growth conditions and the role(s) of each nucleoid protein, we performed for the first time a systematic comparison of the recognition sequence specificity and the DNA-binding affinities among 12 species of the nucleoid protein from E. coli, including the abundant nucleoid proteins in growing E. coli cells, i.e. factor for inversion stimulation (Fis),1 H-NS, HU, IHF, and Lrp, and a set of curved DNA-binding proteins, i.e. CbpA, CbpB, and StpA (6 –15)
Purification of 12 Species of the E. coli DNA-binding Protein—As an initial effort for the systematic comparison of DNA binding properties among the nucleoid proteins in E. coli, we analyzed in this study 12 species of DNA-binding proteins, i.e. CbpA, CbpB, DnaA, Dps, Fis, Hfq, H-NS, HU, IciA, IHF, Lrp, and StpA

Summary

The abbreviations used are

Factor for inversion stimulation; CbpA, curved DNA-binding protein A; CbpB, curved DNA-binding protein B; DnaA, DNA-binding protein A; Dps, DNA-binding protein from starved cells; Hfq, host factor for phage Q␤; H-NS, histone-like nucleoid structuring protein; HU, heat-unstable nucleoid protein; IciA, inhibitor of chromosome initiation A; IHF, integration host factor; Lrp, leucineresponsive regulatory protein; StpA, suppressor of tdϪ phenotype A; bp, base pair(s). 0.75 mM IPTG a Turbidity measured with a Klett-Summerson photometer. Yasuda (Mishima) 2 h This laboratory 1 h R. C. Johnson (Los Angeles) 5 h This laboratory 4 h T. Belfort (Albany) in the stationary phase of cell growth and plays a role in growth-dependent transformation of the nucleoid configuration [4]. Among these 12 proteins, the specificity of DNA recognition has not been reported for Dps, Hfq, IciA, and StpA. The affinity and sequence specificity of DNA binding were examined in parallel for all these 12 species of DNA-binding proteins under the same conditions by gel-mobility shift assays using various DNA probes

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION