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DNA–RNA Interactions Are Critical for Chromosome Condensation in Escherichia Coli

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DNA–RNA Interactions Are Critical for Chromosome Condensation in Escherichia Coli DNA–RNA interactions are critical for chromosome condensation in Escherichia coli Zhong Qiana,1, Victor B. Zhurkinb, and Sankar Adhyaa,1 aLaboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892; and bLaboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 Contributed by Sankar Adhya, September 25, 2017 (sent for review June 23, 2017; reviewed by Yan Jie and Jörg Vogel) Bacterial chromosome (nucleoid) conformation dictates faithful Results regulation of gene transcription. The conformation is condition- Bulged Hairpin Structures Are Critical for the naRNA4 Function in DNA dependent and is guided by several nucleoid-associated pro- Condensation. Atomic force microscopy (AFM) was used to teins (NAPs) and at least one nucleoid-associated noncoding RNA, demonstrate HU and naRNA4-mediated DNA–DNA interac- naRNA4. Here we investigated the molecular mechanism of how tions, a mechanism of chromosome condensation first indicated naRNA4 and the major NAP, HU, acting together organize the by chromosome conformation capture assays (3C) to detect seg- – chromosome structure by establishing multiple DNA DNA contacts mental contacts in chromosomes (12, 15). Previously, we found (DNA condensation). We demonstrate that naRNA4 uniquely acts that naRNA4 may not require extensive homology to target DNA – by forming complexes that may not involve long stretches of DNA in the condensation process (12). Using AFM assays, we de- RNA hybrid. Also, uncommonly, HU, a chromosome-associated pro- termined the required feature(s) of naRNA4 in DNA interactions – tein that is essential in the DNA RNA interactions, is not present in in terms of primary and secondary structures. naRNA4 can form a the final complex. Thus, HU plays a catalytic (chaperone) role in the secondary structure containing two hairpin motifs (Y and Z2), naRNA4-mediated DNA condensation process. each of which contains a mismatched segment (bulge) (Fig. S1). We found that both Y and Z2 motifs are needed; deletion of ei- chromosome structure | DNA–RNA interaction | noncoding RNA | ther one made naRNA4 inactive for DNA condensation (12). Escherichia coli | HU protein Here we constructed several naRNA4 variants and used them in AFM assays to detect DNA–RNA interactions. The following hromosome organization is a focus of intense research in RNA variants were used: (i) RNA without bulges in both hair- Cboth prokaryotic and eukaryotic organisms. It is clear now pins (naRNA4flat); (ii) RNA with two Y hairpins (naRNA4YY); ZZ that chromosomal structures have significant influence on gene (iii) RNA with two Z2 hairpins (naRNA4 ); and (vi) RNA with – ZY transcription (1 3). In transcription regulation, the DNA does the Y and Z2 positions exchanged (naRNA4 ). The predicted sec- not just provide sequence information for binding and action of ondary structures are shown in Fig. S1. We found that, similar to wild- RNA polymerase and transcription factors; the conformation of YY ZZ type naRNA4 that condenses DNA, the naRNA4 ,naRNA4 ,and GENETICS the DNA also plays an important role in faithfully regulating naRNA4ZY constructs were also able to condense DNA. However, transcription of specific genes when appropriate. It has been naRNA4flat without bulges could not make DNA condense under proposed that the Escherichia coli chromosome has a condition- similar conditions (Fig. 1 and Fig. S2 A–L). These results suggest that dependent defined structure (4). A 4.6-Mbp DNA is packaged the naRNA4 constructs must have bulged hairpins for the DNA inside a small cellular volume, and this packaging imparts a condensation function. defined structure to the DNA. Several factors contribute to the DNA packaging. First, the chromosomal DNA is mostly Significance supercoiled, which contributes to its condensation in terms of volume (5, 6). Second, several nucleoid-associated basic pro- This study focuses on the molecular mechanisms of a non- teins (NAPs), such as HU, HNS, CspE, CBP, FIS, etc., par- coding RNA-mediated chromosome organization in Escher- ticipate in chromosome condensation (7). Some of these ichia coli. We show for the first time that noncoding RNA, proteins, such as HU and HNS, modulate gene transcription naRNA4, a critical element at least in part of chromosome specifically as well as globally (1, 8–10). More recently, some structural organization (DNA condensation), may form DNA– noncoding RNAs have also been shown to be involved in RNA complexes in an uncommon way. Surprisingly, we found chromosome structure in E. coli (11, 12). We previously dem- that HU protein catalyzes the DNA–RNA interactions without onstrated that one of them, naRNA4, encoded by a DNA- “ ” REP being part of the final complexes. There are several im- repeat element ( 325), can connect two DNA elements portant examples of noncoding RNA playing critical roles in (dyad symmetry sequences) in the chromosome in the presence eukaryotic chromatin functioning, e.g., heterochromatin as- of HU protein to form DNA loops (12). sembly, gene silencing, and X-inactivation, although nothing Here, we investigated the molecular mechanisms of naRNA4- is known about the nature of DNA–RNA interaction(s) in these mediated chromosome condensation (the product sometimes is processes. Our findings will be of great interest in the chro- referred to as DNA condensate) and the role of HU in the matin field, providing mechanistic insights into the large-scale process. We show that the naRNA4, taking advantage of po- organization of DNA. tential secondary structure, can form DNA–RNA complexes provided that the DNA is supercoiled, converting palindromic Author contributions: Z.Q., V.B.Z., and S.A. designed research; Z.Q. performed research; sequences into cruciform structures leading to chromosome Z.Q., V.B.Z., and S.A. analyzed data; and Z.Q., V.B.Z., and S.A. wrote the paper. condensation in E. coli. We note that naRNA4 is different from Reviewers: Y.J., National University of Singapore; and J.V., University of Würzburg. the long noncoding RNAs (lncRNAs), which are involved in The authors declare no conflict of interest. chromosome structure in the eukaryotic cells (13, 14); lncRNAs Published under the PNAS license. interact with DNA-bound proteins but not directly with DNA. 1To whom correspondence may be addressed. Email: [email protected] or qianz@ Surprisingly, HU protein participating in this process is not a mail.nih.gov. “ ” – part of the final DNA RNA complex, suggesting a chaperon- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. like role of HU. 1073/pnas.1711285114/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1711285114 PNAS | November 14, 2017 | vol. 114 | no. 46 | 12225–12230 Downloaded by guest on October 2, 2021 Note that bacterial DNA has a highly dynamic topology with signal, confirming the existence of DNA in the complexes that relaxed or supercoiled states (16, 17). We observed that negative bind to the antibody (Fig. 2, Top). The results validate the IPP supercoiling of DNA targets is critical for naRNA4-HU–mediated method. We also performed the IPP assays using the naRNA4 DNA condensate formation; no DNA interactions were observed variants (naRNA4flat, naRNA4YY, naRNA4ZZ,andnaRNA4ZY). when relaxed DNA, pSA508rel, was used in the assays (Fig. 1 and Consistent with AFM observations, PCR signals were observed in Fig. S2 N and O). the complexes with naRNA4YY, naRNA4ZZ, and naRNA4ZY but not with naRNA4flat (Fig. 2). We also tested wild-type naRNA4 DNA–naRNA4 Complex Formation Is Critical for DNA Condensation. with relaxed plasmid DNA (pSA508rel)aswellaswild-type Earlier, we proposed various putative models for naRNA4 and naRNA4 and supercoiled plasmid DNA followed by RNaseH HU-mediated DNA–DNA interactions both with and without treatment before IP. Clearly, PCR amplification failed in both formation of DNA–RNA complexes (12). To check if there was cases, suggesting that the complex may contain some kind of any DNA–RNA hybrid formation in the DNA condensate, we DNA–RNA hybrid (Fig. 2). treated the condensates with RNaseH, a sequence nonspecific We further confirmed the above findings about DNA conden- endonuclease that cleaves only RNA in a DNA–RNA hybrid, sation by IP in combination with fluorescence microscopy (IPFM, before AFM imaging. Interestingly, the RNaseH-treated samples flowchart shown in Fig. S3B). We used Cy-3–labeled naRNA4 and did not show any DNA condensation, indicating that the DNA aDNA–RNA hybrid-specific antibody (S9.6), which was coated condensate may contain some form of DNA–RNA hybrid (Fig. 1 with silica beads. In this assay, the beads capturing the potential – and Fig. S2M). DNA RNA complexes showed Cy-3 fluorescence under fluores- To corroborate this observation, we developed an immuno- cence microscopy (FM). Indeed, we observed fluorescent beads in the assays with naRNA4, naRNA4YY, naRNA4ZZ,and precipitation assay in combination with PCR [IP-PCR (IPP) ZY assay] using a specific antibody that could recognize DNA–RNA naRNA4 (Fig. 3). Both our current data and a previous report hybrids, S9.6 (18–20). The protocol is outlined in Fig. S3A. Any (21) confirmed that the silica beads used are free of any auto- DNA–naRNA4 complexes formed would bind to the antibody fluorescence. The frequency of the observed beads with fluores- conjugated to the beads. Using primers specific to the plasmid cent signal among all counted beads varies from 20 to 30% (Table 1). We also detected the Cy-3 signals in assays with naRNA4 and DNA, we could amplify DNA fragments in the eluates from the YY ZZ ZY beads. An amplification of the DNA signal would indicate the its variants (naRNA4 ,naRNA4 ,andnaRNA4 )inthe absence of HU protein at 2–5% levels, which are reproducibly existence of DNA–RNA complexes in the condensates. These lower than the frequency observed in the presence of HU (Fig. 3 results with the wild-type naRNA4 in the presence of plasmid and Table 1).
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