RNA-binding as chaperones of DNA binding proteins from starved cells

https://doi.org/10.1016/j.bbrc.2020.01.121Get rights and content

Highlights

  • RNA maintains the solubility and stability of Dps in the absence of DNA.

  • RNA preempts Dps from binding to DNA in the exponential phase.

  • In the stationary phase, RNA degradation frees Dps for binding to chromosomal DNA.

  • RNA serves as a ‘holdase’ type chaperone for the functional competence of Dps.

Abstract

DNA-binding proteins from starved cells (Dps) in Escherichia coli protects DNA from multiple stresses during the stationary phase by forming a stable Dps-DNA complex. In contrast, Dps cannot bind to DNA during the exponential phase and it has not been clear why Dps conditionally binds to DNA depending on the growth phase. In this study, we show that DNA-free Dps in the exponential phase can also bind to RNA and the preemptive binding of RNA precludes DNA from interacting with Dps. The critical role of RNA in modulating the stability and functional competence of Dps and their morphology, leads us to propose a two-state model of Dps in executing stress responses. In the exponential phase, Dps is present predominantly as ribonucleoprotein complex. Under starvation, RNAs are degraded by up-regulated RNases, activating Dps to bind with chromosomal DNAs protecting them from diverse stresses. A dual role of RNA as an inhibitor of DNA binding and chaperone to keep dynamic functional status of Dps would be crucial for operating an immediate protection of chromosomal DNAs on starvation. The holdase-type chaperoning role of RNA in Dps-mediated stress responses would shed light on the role of RNAs as chaperone (Chaperna).

Introduction

Bacteria are constantly faced with environmental changes and have evolved to adapt to a variety of stresses. Under starved condition in the stationary phase, Escherichia coli (E. coli) cells accumulate DNA-binding proteins from starved cells (Dps) to protect their genomic DNAs non-specifically [1]. Whereas the amount of Dps is low in the exponential phase, its concentration dramatically increases (up to twelve times) during the stationary phase, becoming predominant among nucleoid-associated proteins [2]. Under this condition, Dps keeps DNA as a compacted form to avoid stresses, such as oxidative cleavage, low pH, and over-exposure to iron [3,4].

The Dps assembles into a spherical dodecameric structure with a hollow core. The overall surface charge of Dps is mostly negative and repulsive to DNA binding [4] and does not display any known DNA-binding motif [5]. The lysine residue on N-terminal extended region of Dps (Dps-N) is crucial for self-aggregation as well as for Dps-driven DNA condensation [3]. The deletion of this portion of the N-terminus or substitution mutations of key lysine residues in Dps-N impairs or eliminates the condensation activity [3,4].

Both Dps and bacterioferritin (Bfr) belong to the ferritin superfamily and are considered as mini-type and maxi-type ferritin because they are assembled of 12 and 24 monomers, respectively [6]. The structures of both proteins are highly conserved except for the N-terminal residue: Dps has an extended region as intrinsically disordered region (IDR) whereas Bfr does not [6]. Such biophysical differences between the two closely related proteins suggest that the understanding of the stress-response function of Dps could be deduced from previous studies in Bfr. The activity of Bfr-conjugated proteins could be greatly enhanced by fusion to an IDR containing RNA-binding modules; variety of protein domains, either antigens or antibody fragments, were successfully displayed and assembled on the surface of chimeric Bfr [7,8]. Potent stimulation of the solubility of Bfr and the kinetic pathways in favor of the folding and assemblage of Bfr nanostructure was mediated by interaction between RNAs and IDRs [7,8]. In this regard, we asked if the disordered Dps-N also interacts with RNAs as it binds to DNAs, contributing to its in-cell solubility and stress responses [3,6].

In this study, we showed, for the first time, that RNA interacts with E. coli Dps, maintaining the stability and functional competence of Dps in the exponential phase. The preemptive binding with RNA precludes Dps from binding with chromosomal DNA in the pre-stationary phase providing a ‘holdase’ type chaperoning role of RNA in Dps-mediated stress responses [9].

Section snippets

Expression vector construction & protein expression

The genomic DNA of E. coli BL21(DE3) was extracted by AccuPrep® (Bioneer) and PCR was performed for generation of Dps, and histidine tagged form (Dps-His), and mutated Dps (3mDps; K5/8/10A) with specific primer (Table S1). The genes were inserted into the pGEpBAD vector, which were derived from pGEMEX-1 (promega) and pET-43.1a (+) (Novagen). E. coli BL21(DE3) was transformed with each expression vector, and the transformed cells were grown in 3 mL of LB medium with ampicillin (50 μg/mL)

Dps depended on the presence of RNAs

In order to investigate potential factors that affect the stability of Dps in E. coli, we extracted lysates from Dps over-expressing E. coli cells in the exponential phase. To preserve the physical integrity of Dps complex without DNAs, we employed mild lysis with DNase I treatment instead of the conventional sonication method. Even though Dps was free from DNAs, soluble Dps was remained in supernatant fraction (S of Fig. 1). Based on the fact that RNA manages stability and activity of the

Conclusion

Elucidating a novel mechanism of RNAs on Dps-mediated stress responses, the report further shed lights on the role of RNAs as chaperones in protein homeostasis [19]. The chaperonic role of RNAs identified in this study resembles the ‘holdase’ type of molecular chaperones [20,21]. It has been reported that RNAs can play a role in assisting protein folding like molecular chaperones in both physiological and pathological conditions [22]. A novel function of RNA as chaperone was described as

Author contributions

CP designed experiments and schematic models; CP and YJ purified candidate proteins and performed experiments; YJ optimized protein purification condition; CP, YJ, YJK, HJ, and BLS wrote the manuscript. All authors read and revised the manuscript.

Acknowledgements

This study was supported by the Bio & Medical Technology Development Program of the National Research Foundation (NRF) (No. NRF-2018M3A9H4079358).

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