期刊：Current Genetics（2.695/Q3）
DNA repeat sequences: diversity and versatility of functions (2016)
DNA重复序列：功能的多样性和多功能性
Abstract Although discovered decades ago, the molecular identification, the diversity and versatility of functions, and the evolutionary origin of repeat DNA sequences (REPs) containing palindromic units in prokaryotes are now bringing attention to a wide range of biological scientists. A brief account of the current state of the repeat DNA sequences is presented here.
Keywords Palindromic units · Repeat DNA sequence · naRNA · BIME · REP
摘要 虽然早在几十年前就被发现，但原核生物中含有回文单元的重复 DNA 序列（REPs）的分子鉴定、功能的多样性和多功能性以及进化起源现在引起了广泛的生物科学家的关注。 此处简要介绍了重复 DNA 序列的当前状态。
关键词 回文单位 · 重复 DNA 序列 · naRNA · BIME · REP
Introduction
Three decades ago genetic analysis of prokaryotic chromosomes identified the existence of large numbers of sequences called repetitive extra-genic palindromes (REPs) between genes (ORFs) long before repetitive “junk” sequences were discovered in eukaryotes (Higgins et al. 1982; Lee et al. 2004; Tobes and Ramos 2005). Although remained “mystic” for years, more researchers are now realizing that the so-called repeats or junk sequences often participate in diverse cellular functions including gene evolution, gene expression, mRNA stabilization, gene organization, gene mobility, cellular immunity against foreign genes, as well as in gene engineering in both prokaryotes and eukaryotes (Aranda-Olmedo et al. 2002; Bachellier et al. 1993; George et al. 2015; Higgins et al. 1982; Rocco et al. 2010; Tobes and Pareja 2005; Tobes and Ramos 2005). Our understanding of the role of specific repeat sequences has not yet been fully realized, and extensive investigations are now being pursued in this exciting subject. Thus, a synopsis on the history of the diversity and versatility of functions of the repeated DNA sequences and their significance in cell behavior may be useful at this juncture. The REP element functions discussed here and the references thereof are arbitrary, not exhaustive, and only to make some points. More recent identification of a new class of short palindromic repeats, known as clustered regularly interspaced short palindromic repeats (CRISPR) related to imparting immunity against foreign DNA invasion in prokaryotes (Barrangou et al. 2007; Bondy-Denomy et al. 2015; Garneau et al. 2010; Horvath and Barrangou 2010), is another milestone in the area of DNA repeat sequences. CRISPR has been the subject of several excellent reviews (Sternberg et al. 2016), and is not covered here.
30 年前，原核生物染色体的遗传分析确定了在真核生物中发现重复“垃圾”序列之前很久，在基因 (ORF) 之间存在大量称为重复基因外回文 (REP) 的序列 (Higgins et al. 1982; Lee et al. al. 2004；Tobes 和 Ramos 2005）。尽管多年来一直“神秘”，但越来越多的研究人员现在意识到，所谓的重复序列或垃圾序列通常参与多种细胞功能，包括基因进化、基因表达、mRNA 稳定、基因组织、基因迁移、针对外源基因的细胞免疫、以及原核生物和真核生物的基因工程（Aranda-Olmedo et al. 2002; Bachellier et al. 1993; George et al. 2015; Higgins et al. 1982; Rocco et al. 2010; Tobes and Pareja 2005; Tobes和拉莫斯 2005）。我们对特定重复序列的作用的理解尚未完全实现，目前正在对这个令人兴奋的主题进行广泛的调查。因此，关于重复 DNA 序列功能的多样性和多功能性的历史及其在细胞行为中的意义的概要可能在此时有用。这里讨论的 REP 元素功能及其引用是任意的，并非详尽无遗，仅用于说明一些观点。最近发现了一类新的短回文重复序列，称为成簇的规则间隔短回文重复序列 (CRISPR)，与原核生物对外源 DNA 入侵的免疫有关（Barrangou 等人，2007；Bondy-Denomy 等人，2015；Garneau 等人） al. 2010; Horvath and Barrangou 2010)，是 DNA 重复序列领域的另一个里程碑。 CRISPR 已成为几篇优秀评论的主题（Sternberg 等人，2016 年），此处未涉及。
Higgins et al., first described several hundred base-pair long homologous genetic elements that were present in extra-cistronic regions of some operons of different bacterial species. The elements contained G:C-rich imperfect dyad symmetry sequences called palindromic units (PUs) which are capable of forming cruciform structures in DNA (stem and loop structures in RNA, if transcribed) (Higgins et al. 1982). They also reported the occurrences of PUs both within (inter-cistronic) and outside (inter-operonic) ORF-encoding regions with 1-3 tandem repeats. Further genome wide surveys revealed that the PUs also exist in the form of a complex higher order organization, termed as Bacterial Interspersed Mosaic Elements (BIMEs), containing a number of other conserved sequences (Gilson et al. 1991a, b). Such complex BIMEs may contain PU units in inverse orientations separated by a linker (l) sequence (Messing et al. 2012). For example, there are about 600 copies of BIMEs in Escherichia coli, and about 1600 in Stenotrophomonas maltopholia (Bachellier et al. 1999; Dimri et al. 1992; Gilson et al. 1991a; Rocco et al. 2010). Extensive studies on BIMEs have been carried out with respect to their organization, species distribution, and functions. The components of BIMEs include PUs of three different motifs (Y, Z 1 and Z2, see Fig. 1), terminal conserved sequences (A and B), internal motifs (L and S) and left terminal motifs (l, r and s) in BIME-1 and BIME-2 (Fig. 2) (Bachellier et al. 1994; Gilson et al. 1991b). An interesting property of REPs is their functional diversity. The following discoveries with the REP elements led to identification of some of their amazingly diverse functions.
Higgins 等人首先描述了数百个碱基对长的同源遗传元件，这些元件存在于不同细菌物种的一些操纵子的顺反子外区域。这些元素包含富含 G:C 的不完美二元对称序列，称为回文单元 (PU)，它们能够在 DNA 中形成十字形结构（RNA 中的茎和环结构，如果转录的话）（Higgins 等人，1982 年）。他们还报告了在（顺反子间）和外部（operonic 间）ORF 编码区域内出现 1-3 个串联重复的 PU。进一步的全基因组调查显示，PU 还以复杂的高阶组织形式存在，称为细菌散布镶嵌元件 (BIME)，包含许多其他保守序列 (Gilson et al. 1991a, b)。这种复杂的 BIME 可能包含反向方向的 PU 单元，由接头 (l) 序列分隔 (Messing et al. 2012)。例如，大肠杆菌中有大约 600 个 BIME 拷贝，麦芽窄食单胞菌中有大约 1600 个（Bachellier 等人 1999；Dimri 等人 1992；Gilson 等人 1991a；Rocco 等人 2010）。已经对 BIME 的组织、物种分布和功能进行了广泛的研究。 BIME 的成分包括三个不同基序的 PU（Y、Z 1 和 Z2，见图 1）、末端保守序列（A 和 B）、内部基序（L 和 S）和左末端基序（l、r 和 s ) 在 BIME-1 和 BIME-2 (图 2) (Bachellier et al. 1994; Gilson et al. 1991b)。 REP 的一个有趣特性是它们的功能多样性。 REP 元素的以下发现导致了它们的一些惊人的多样化功能的识别。
Fig. 1
Fig. 1 Sequences of three PU motifs (Y, Z1 and Z2) from BIME-1 and BIME-2 in E. coli (Bachellier et al. 1999). The dyad symmetry sequences are boxed. The three motifs are homologous but not identical. Thus a schematic representation of the PU units is shown below by a solid triangle (black) to signify the relative orientations of each PU unit
图 1 来自 BIME-1 和 BIME-2 的三个 PU 基序（Y、Z1 和 Z2）在大肠杆菌中的序列（Bachellier et al. 1999）。 对偶对称序列加框。 这三个基序是同源的，但并不完全相同。 因此，PU 单元的示意图如下所示，由实心三角形（黑色）表示，以表示每个 PU 单元的相对方向
Fig. 2 A schematic representation of BIME-1 and BIME-2 in E. coli. Solid triangles show the orientation from head to tail. BIME-1 contains a Y motif and a Z 1 motif which are head to head and connected by a L motif. Two external motifs (A and B) could be located at the tails of Y and Z1 motifs, respectively. BIME-2 contains ‘n’ repeats, n being 1–6, of Y and Z 2 motifs connected by an internal S motif. Another sequence motif (l, r or s) is located at the tail of Z2
图 2 大肠杆菌中 BIME-1 和 BIME-2 的示意图。 实心三角形显示从头到尾的方向。 BIME-1 包含一个 Y 基序和一个 Z 1 基序，它们头对头并由一个 L 基序连接。 两个外部基序（A 和 B）可以分别位于 Y 和 Z1 基序的尾部。 BIME-2 包含由内部 S 基序连接的 Y 和 Z 2 基序的“n”个重复，n 为 1-6。 另一个序列基序（l、r 或 s）位于 Z2 的尾部
REP sequences binding to proteins
DNA polymerase I binding
Since the discovery of REP elements in bacteria, their functions were speculative until the publications from Maurice Hofnung’s laboratory (Gilson et al. 1984, 1986; Higgins et al. 1982) showing that a protein present in nucleoid (called “chromoid” at the time) extract protected REP DNA, e.g. in the intergenic region of malE/malF and lamB/malM (named as interE and interB, respectively) from digestion by exonuclease III (Gilson et al. 1986). Gilson et al., identified the responsible protein for REP protection to be a DNA repair enzyme—an intact DNA polymerase I (Gilson et al. 1990).
与蛋白质结合的 REP 序列
DNA聚合酶I结合
自从在细菌中发现 REP 元素以来，它们的功能一直是推测性的，直到 Maurice Hofnung 实验室的出版物 (Gilson et al. 1984, 1986; Higgins et al. 1982) 表明存在于类核中的蛋白质（当时称为“染色质” ) 提取受保护的 REP DNA，例如 在由外切核酸酶 III 消化产生的 malE/malF 和 lamB/malM（分别命名为 interE 和 interB）的基因间区域中（Gilson 等，1986）。 Gilson 等人确定了负责保护 REP 的蛋白质是一种 DNA 修复酶——一种完整的 DNA 聚合酶 I（Gilson 等人 1990）。
IHF binding
IHF (Integration host factor) was identified for its role in the site-specific integration of temperate bacteriophage λ to the host chromosome. It acts by binding to a specific DNA sequence: 5′-PyAANNNPuTTGATW-3′ at the λ DNA attachment site. IHF binding sequences (Ihf) are frequently located between two PU sequences: PU-Ihf-PU (Boccard and Prentki 1993; Oppenheim et al. 1993; Rudd 1998). The PU-Ihf-PU sequence arrangement called RIP by one group (Oppenheim et al. 1993) and RIB by another (Boccard and Prentki 1993) occurs 79 times around the chromosome of E. coli (Rudd 1998). The structure of RIP/RIB elements is shown in Fig. 3. The 79 RIP/RIB elements are a subset of 355 REP elements (Rudd 1998). Different from the other 276 REP elements, RIP/RIB elements binds to IHF (Boccard and Prentki 1993; Oppenheim et al. 1993). These sequences are also found in the chromosome of other closely related enteric bacteria. For example, Bachellier et al., reported the existence of a BIME element, which is composed of two convergent REP-like units flanking an Ihf site, located at the 3′ end of pulA gene in Klebsiella aerogenes (Bachellier et al. 1993). The functions of these sequences have been proposed to be (i) participation in chromosome condensation (Oppenheim et al. 1993) and (ii) helping DNA gyrase binding (Boccard and Prentki 1993) (see below).
IHF（整合宿主因子）因其在温带噬菌体 λ 与宿主染色体的位点特异性整合中的作用而被鉴定。它通过结合特定的 DNA 序列发挥作用：5'-PyAANNNPuTTGATW-3' 在 λ DNA 附着位点。 IHF 结合序列 (Ihf) 经常位于两个 PU 序列之间：PU-Ihf-PU (Boccard and Prentki 1993; Oppenheim et al. 1993; Rudd 1998)。一组称为 RIP (Oppenheim et al. 1993) 和另一组称为 RIB (Boccard 和 Prentki 1993) 的 PU-Ihf-PU 序列排列在大肠杆菌的染色体周围出现了 79 次 (Rudd 1998)。 RIP/RIB 元素的结构如图 3 所示。79 个 RIP/RIB 元素是 355 个 REP 元素的子集（Rudd 1998）。与其他 276 个 REP 元素不同，RIP/RIB 元素与 IHF 结合（Boccard 和 Prentki 1993；Oppenheim 等人 1993）。这些序列也存在于其他密切相关的肠道细菌的染色体中。例如，Bachellier 等人报道了 BIME 元件的存在，该元件由位于产气克雷伯菌 pulA 基因 3' 端的 Ihf 位点两侧的两个会聚 REP 样单元组成（Bachellier 等人，1993） .已经提出这些序列的功能是（i）参与染色体凝聚（Oppenheim et al. 1993）和（ii）帮助 DNA 促旋酶结合（Boccard 和 Prentki 1993）（见下文）。
Fig. 3 A typical structure of a RIB/RIP element (Boccard and Prentki 1993). A RIP/RIB element contains PU motifs flanked by an L motif with a functional IHF binding site
图 3 RIB/RIP 元素的典型结构（Boccard 和 Prentki 1993）。 RIP/RIB 元件包含 PU 基序，其两侧是具有功能性 IHF 结合位点的 L 基序
DNA gyrase binding
Bacterial DNA gyrase is a class II topoisomerase, which alters the topology of DNA by passing a segment of it through a double stranded break in another segment of the same circular DNA molecule. By this mechanism, DNA gyrase introduces negative supercoiling to DNA to relieve the torsional stress arising from transcription and replication complexes (Liu and Wang 1987; Maxwell and Gellert 1986). Yang and Ames showed that DNA segments containing a natural REP element with two PUs bind to gyrase to form DNA/gyrase complexes (Yang and Ames 1988). Espeli and Boccard demonstrated in vivo cleavage of E. coli BIME-2 repeats by DNA gyrase in the presence of oxolinic acid (Espeli and Boccard 1997). The property of oxolinic acid, which is an inhibitor of gyrase action but not of DNA binding, also helped identification of sites of gyrase binding sites in the E. coli chromosome (Morrison and Cozzarelli 1979). It was observed that the frequency of DNA gyrase mediated cuts varies from site to site, and DNA gyrase cut target DNA once every two REPs. They identified a single cleavage site in the Y motif and a central 44 bp fragment as the target of gyrase (Espeli and Boccard 1997).
DNA 促旋酶结合
细菌 DNA 促旋酶是一种 II 类拓扑异构酶，它通过使 DNA 的一个片段通过同一环状 DNA 分子的另一个片段中的双链断裂来改变 DNA 的拓扑结构。通过这种机制，DNA 促旋酶将负超螺旋引入 DNA，以减轻由转录和复制复合物产生的扭转应力（Liu 和 Wang 1987；Maxwell 和 Gellert 1986）。 Yang 和 Ames 表明，含有一个天然 REP 元件和两个 PU 的 DNA 片段与旋转酶结合形成 DNA/旋转酶复合物（Yang 和 Ames 1988）。 Espeli 和 Boccard 证明了在体内存在 oxolinic 酸的情况下，大肠杆菌 BIME-2 重复序列可通过 DNA 促旋酶进行切割（Espeli 和 Boccard 1997）。 oxolinic acid 是促旋酶作用的抑制剂，但不抑制 DNA 结合，其特性也有助于识别大肠杆菌染色体中促旋酶结合位点的位点（Morrison 和 Cozzarelli 1979）。据观察，DNA 促旋酶介导的切割频率因位点而异，DNA 促旋酶每两个 REP 切割一次目标 DNA。他们确定了 Y 基序中的一个切割位点和一个中央 44 bp 片段作为旋转酶的靶标（Espeli 和 Boccard 1997）。
DNA transposase binding
DNA transposons or transposable elements are mobile genetic elements that are able to move or spread to other locations in genomes. These actions are called transposition and are catalyzed by DNA transposases (Hickman and Dyda 2015). The simplest example of a transposon is specific insertion sequences (IS elements), that can transfer from one chromosomal location to another (Nunvar et al. 2010). They belong to a family of sequences ~700–1500 bp long that contains one or more ORFs, which are flanked by two identical sequences in opposite orientations. One of the ORFs encodes the protein transposes. DNA transposase binds to the terminal inverted repeats, catalyze DNA cleavage and insertional recombination at another site (Chandler et al. 2013; Hickman and Dyda 2015; Nunvar et al. 2010; Ton-Hoang et al. 2012). IS elements are found both in bacteria and archaea (Filee et al. 2007; Florek et al. 2014; Lam and Roth 1983).
DNA转座酶结合
DNA转座子或转座因子是能够移动或扩散到基因组中其他位置的移动遗传元件。 这些行为称为转座，由 DNA 转座酶催化（Hickman 和 Dyda 2015）。 转座子最简单的例子是特定的插入序列（IS 元件），它可以从一个染色体位置转移到另一个位置（Nunvar 等人，2010）。 它们属于一个长约 700-1500 bp 的序列家族，其中包含一个或多个 ORF，其两侧是两个方向相反的相同序列。 其中一个 ORF 编码蛋白质转座。 DNA 转座酶与末端反向重复序列结合，在另一个位点催化 DNA 切割和插入重组（Chandler 等人 2013；Hickman 和 Dyda 2015；Nunvar 等人 2010；Ton-Hoang 等人 2012）。 IS 元素存在于细菌和古生菌中（Filee et al. 2007; Florek et al. 2014; Lam and Roth 1983）。
Functions of REP elements
Serving as transcription terminators
As mentioned, the REP elements appear to have various functions. One of the early perceived roles of REPs is helping transcription termination at the end of an operon or reduction of transcription within an operon to reduce of expression of distal genes (natural polarity). Previously, transcription termination at specific DNA sites were thought of two kinds. Type I: Sequence dependent intrinsic signal that generates various forms of stem-loop RNA structures in the elongating RNA that make RNA polymerase to stop. Type II: Nascent RNA Sequences of certain composition (Cytosine-rich) with the help of proteins, like Rho, make RNA polymerase to terminate transcription. It has been demonstrated that the presence of REP elements within the inter-cistronic regions in several operons reduces distal transcription causing natural polarity but needs the termination factor Rho to be effective. Mutational analysis showed that Rho action in these cases needs a conserved sequence embedded in a PU: WWNGCCKNATNMGGCNWW. Thus REP elements together with Rho are involved in natural polarity. How Rho reduces transcription at this sequence, and if this termination is also modulated is not known. (Espeli et al. 2001). Since the inter-cistronic occurrences of REPs are not universal in operons, their evolutionary origin in some operons has been attributed to the biological need of natural polarity in those.
REP 元件的功能
作为转录终止符
如前所述，REP 元件似乎具有各种功能。 REPs 的早期感知作用之一是帮助在操纵子末端终止转录或减少操纵子内的转录以减少远端基因的表达（自然极性）。以前，在特定 DNA 位点的转录终止被认为有两种。 I 型：序列依赖性内在信号，在延长 RNA 中产生各种形式的茎环 RNA 结构，使 RNA 聚合酶停止。 II 型：某些组成（富含胞嘧啶）的新生 RNA 序列在蛋白质（如 Rho）的帮助下，使 RNA 聚合酶终止转录。已经证明，在几个操纵子的顺反子间区域内 REP 元件的存在减少了导致自然极性的远端转录，但需要终止因子 Rho 才能有效。突变分析表明，在这些情况下，Rho 作用需要嵌入 PU 中的保守序列：WWNGCCKNATNMGGCNWW。因此，REP 元素与 Rho 一起参与自然极性。 Rho 如何减少该序列的转录，以及该终止是否也被调节尚不清楚。 （Espeli 等人，2001 年）。由于 REP 的顺反子间发生在操纵子中并不普遍，因此它们在某些操纵子中的进化起源已归因于这些操纵子中自然极性的生物学需要。
Toll‑like receptor recognizing REP elements
Toll-like receptors (TLRs) are conserved transmembrane proteins containing the binding sites for both their ligands and their co-receptors in mammalian cells. The function of TLRs is to recognize specific ligands to initiate inflammatory processes, thus activating signaling molecules to promote microglial phagocytosis, cytokine release and the expression of the co-stimulatory molecules needed for adaptive immune responses. In mammals, a total of twelve types of TLRs have been detected, in human, there are nine, termed as TLRs 1–9 (Hanke and Kielian 2011; Parthiban and Mahendra 2015). A very different role of bacterial REP elements is to induce inflammatory process in mammalian systems. Hemmi et al., showed that mammal cellular immuno-stimulatory response to bacterial DNA is mediated by TLR9. TLR9 could distinguish bacterial DNA from self DNA (Hemmi et al. 2000). Magnusson et al., searched for bacterial DNA sequences as natural TLR9 ligands. By using synthetic REP DNA from different bacteria wrapped in lipofectin, the authors detected the induction pro-inflammatory cytokine, IFN-α. This ability of REPs was totally dependent on TLR9 signaling because splenocytes from TLR9 knockout mice did not produce IFN-α when stimulated with REPs, demonstrating that REP elements are natural TLR9 ligands which allow the innate immune system to distinguish bacterial DNA (Magnusson et al. 2007).
识别 REP 元件的 Toll 样受体
Toll 样受体 (TLR) 是保守的跨膜蛋白，在哺乳动物细胞中含有它们的配体和共受体的结合位点。 TLR 的功能是识别特定配体以启动炎症过程，从而激活信号分子以促进小胶质细胞吞噬、细胞因子释放和适应性免疫反应所需的共刺激分子的表达。在哺乳动物中，总共检测到 12 种 TLR，在人类中，有 9 种，称为 TLR 1-9（Hanke 和 Kielian 2011；Parthiban 和 Mahendra 2015）。细菌 REP 元件的一个非常不同的作用是在哺乳动物系统中诱导炎症过程。 Hemmi 等人表明，哺乳动物细胞对细菌 DNA 的免疫刺激反应是由 TLR9 介导的。 TLR9 可以区分细菌 DNA 和自身 DNA (Hemmi et al. 2000)。 Magnusson 等人搜索了作为天然 TLR9 配体的细菌 DNA 序列。通过使用包裹在 lipofectin 中的不同细菌的合成 REP DNA，作者检测到了诱导性促炎细胞因子 IFN-α。 REPs 的这种能力完全依赖于 TLR9 信号传导，因为来自 TLR9 敲除小鼠的脾细胞在用 REPs 刺激时不会产生 IFN-α，这表明 REP 元件是天然的 TLR9 配体，它允许先天免疫系统区分细菌 DNA（Magnusson 等人，2013 年）。 2007）。
Participating in nucleoid DNA folding
Despite several significant developments in our understanding of bacterial chromosome (nucleoid) structure, the field still remains a challenging subject. Up to date, several protein components of the nucleoid have been identified that result in specific structures of the corresponding nucleoid protein bound DNA complexes (Ishihama 2009). Although a 3-D structural arrangement of the nucleoid DNA has been proposed that relates to transcription (Kar et al. 2005; Macvanin and Adhya 2012), a comprehensive and credible answer to this proposal must await identification of all the critical elements and principles of their participation in the DNA organization and their effect on gene transcription. Besides the protein factors, the involvement of REP elements as specific DNA sequences in nucleoid condensation has also been suggested (Qian et al. 2015). BIME may participate in chromosome condensation at several levels. The role of REP elements in chromosome architecture is discussed below.
参与类核 DNA 折叠
尽管我们对细菌染色体（类核）结构的理解取得了一些重大进展，但该领域仍然是一个具有挑战性的课题。迄今为止，已鉴定出类核的几种蛋白质成分，它们导致相应的类核蛋白结合 DNA 复合物的特定结构（Ishihama 2009）。尽管已经提出了与转录相关的类核 DNA 的 3-D 结构排列（Kar et al. 2005；Macvanin 和 Adhya 2012），但对该提议的全面和可信的回答必须等待所有关键要素和原则的确定。它们参与 DNA 组织及其对基因转录的影响。除了蛋白质因素外，还提出了 REP 元件作为特定 DNA 序列参与类核浓缩（Qian et al. 2015）。 BIME 可能在多个层面参与染色体凝聚。下面讨论 REP 元件在染色体结构中的作用。
DNA polymerase I
Although interaction of REP elements with DNA polymerase I has been demonstrated, no functional assignments have yet been made to this interaction. A potential role in DNA repair is anticipated, an additional role in DNA condensation has not been ruled out.
DNA聚合酶I
尽管已经证明了 REP 元件与 DNA 聚合酶 I 的相互作用，但尚未对这种相互作用进行功能分配。 预计在 DNA 修复中的潜在作用，尚未排除在 DNA 凝聚中的额外作用。
IHF
It binds to a specific RIB/RIP sequence. Since IHF binding bends DNA considerably, the DNA bent points must help DNA condensation.
它绑定到特定的 RIB/RIP 序列。 由于 IHF 结合大大弯曲 DNA，DNA 弯曲点必须有助于 DNA 凝聚。
DNA gyrase
The average negative superhelical density in the E. coli chromosome created by is 0.06 (Hatfield and Benham 2002). This amount of superhelicity compacts the 4.6 MB circular DNA by 10 3-fold (Postow et al. 2004). The DNA gyrase binding sites are embedded in a two-PU unit REP element located around the chromosome.
DNA旋转酶
大肠杆菌染色体中的平均负超螺旋密度为 0.06（Hatfield 和 Benham 2002）。 这种超螺旋量将 4.6 MB 环状 DNA 压缩了 10 3 倍（Postow 等人，2004）。 DNA 促旋酶结合位点嵌入位于染色体周围的两个 PU 单元 REP 元件中。
DNA transposes
DNA transposes action initiates after its binding to REP DNA sequences. DNA transposition does not directly condense DNA but participates in chromosome re-arrangements by translocating DNA segments from one locus to another or inverting large segments of the chromosome.
DNA转座
DNA 转座作用在其与 REP DNA 序列结合后开始。 DNA转座不直接浓缩DNA，而是通过将DNA片段从一个基因座转移到另一个基因座或反转染色体的大片段来参与染色体重排。
HU
The nucleoid protein HU binds at high efficiency to PU units (Arthanari et al. 2004), which in a supercoiled state assumes a cruciform structure, and bends DNA. Bending would help DNA compaction as IHF does. Besides specific bindings, this most abundant nucleoid protein also induces specific architectural twist to DNA as well as brings different DNA segments together (bunching) by binding non-specifically (Hammel et al. 2016). This property contributes to DNA compaction in a major way.
类核蛋白 HU 与 PU 单元高效结合 (Arthanari et al. 2004)，在超螺旋状态下呈现十字形结构并弯曲 DNA。弯曲会像 IHF 一样帮助 DNA 压实。除了特异性结合之外，这种最丰富的类核蛋白还可以诱导 DNA 的特定结构扭曲，并通过非特异性结合将不同的 DNA 片段聚集在一起（聚束）（Hammel 等人，2016 年）。这种特性在很大程度上有助于 DNA 压缩。
non‑coding RNA (naRNA)
A completely different kind of DNA compaction mediated by cruciform DNA structures has been discovered recently that involve both HU and the nucleoid associated non-coding RNA (naRNA4) (Qian et al. 2015), or nc5 which has been recently identified by RIP-Chip assays by its ability to bind to HU (Macvanin et al. 2012). HU binding to nc5 or naRNA4 was confirmed by the EMSA assay (Macvanin et al. 2012; Qian et al. 2015). naRNA4 has been shown to be involved in nucleoid structural condensation. It has been suggested that naRNA4 collaborates with HU in chromosome condensation. (Qian et al. 2015). Transmission electron microscopy (TEM) analysis of cells deleted for genes encoding the naRNA4 and/or HU showed de-condensed nucleoid compared to that in wild type cells consistent with the idea that naRNA4 complexed with HU participates in the compaction and maintenance of the nucleoid structure (Macvanin and Adhya 2012; Qian et al. 2015). It is interesting that the naRNA4 is encoded by a BIME element (BIME-2) composed of two PUs (Z 2 and Y). Further confirmation of the nucleoid condensation mediated by HU and naRNA4 came from atomic force microscopy (AFM) that clearly showed distal DNA connections in the presence of both naRNA4 and HU (Qian et al. 2015). naRNA4/HU complexes that bring together two DNA segments containing PUs, which are in cruciform structures in a supercoiled DNA. Based on in vivo or in vitro data, alternate mechanisms for interactions between two DNA cruciforms mediated by HU and naRNA4 have been proposed (Fig. 4). Although the molecular details of the DNA–DNA bridging by naRNA4 and HU remain to be investigated, the involvement of a specific non-coding RNA in nucleoid condensation in collaboration with a nucleoid protein was previously unknown. Analysis of the online available RNAseq data (Raghavan et al. 2011) showed that of the 355 REP elements present in E. coli chromosome, 252 are transcribed. It would not be surprising that RNA products of other BIMEs may also be involved in chromosome condensation. The mechanism by which BIME products condense chromosome has been revealed by the use of Chromosome Confirmation Capture (3C) analysis. It has been reported that out of 253 possible combinations of BIME elements around the E. coli chromosome, 27 pairs show positive connections to each other. 3C analysis also demonstrated that some of the connections among BIMEs, are not HU and naRNA4 dependent; other RNAs or proteins may be involved in making the other intra-chromosomal connections not dependent upon HU or naRNA4 (Qian et al. 2015). This non-coding RNA mediated DNA compaction compares to the role of ncRNAs in gene repression in hetero-chromatins in eukaryotes (Jorgensen 1990; Yang and Li 2016).
非编码 RNA (naRNA)
最近发现了一种完全不同类型的由十字形 DNA 结构介导的 DNA 压实，它涉及 HU 和类核相关的非编码 RNA (naRNA4) (Qian et al. 2015)，或最近由 RIP-Chip 鉴定的 nc5通过其与 HU 结合的能力进行分析 (Macvanin et al. 2012)。 EMSA 测定证实了 HU 与 nc5 或 naRNA4 的结合（Macvanin 等人 2012；Qian 等人 2015）。已显示 naRNA4 参与类核结构凝聚。有人提出 naRNA4 与 HU 在染色体凝聚中协作。 （钱等人，2015）。对编码 naRNA4 和/或 HU 的基因缺失的细胞的透射电子显微镜 (TEM) 分析显示，与野生型细胞相比，类核被去浓缩，这与与 HU 复合的 naRNA4 参与类核结构的压实和维持的观点一致（Macvanin 和 Adhya 2012；Qian 等人 2015）。有趣的是，naRNA4 是由一个由两个 PU（Z 2 和 Y）组成的 BIME 元素（BIME-2）编码的。由 HU 和 naRNA4 介导的类核凝聚的进一步确认来自原子力显微镜 (AFM)，该显微镜清楚地显示了在 naRNA4 和 HU 存在的情况下远端 DNA 连接 (Qian et al. 2015)。 naRNA4/HU 复合物将两个含有 PU 的 DNA 片段结合在一起，这些片段在超螺旋 DNA 中呈十字形结构。基于体内或体外数据，已经提出了由 HU 和 naRNA4 介导的两个 DNA 十字形之间相互作用的替代机制（图 4）。尽管由 naRNA4 和 HU 桥接的 DNA-DNA 的分子细节仍有待研究，但特定的非编码 RNA 与类核蛋白协作参与类核凝聚的过程以前是未知的。在线可用的 RNAseq 数据分析 (Raghavan et al. 2011) 表明，在大肠杆菌染色体中存在的 355 个 REP 元素中，有 252 个被转录。其他 BIME 的 RNA 产物也可能参与染色体凝聚也就不足为奇了。使用染色体确认捕获 (3C) 分析揭示了 BIME 产品凝聚染色体的机制。据报道，在大肠杆菌染色体周围的 253 种可能的 BIME 元素组合中，有 27 对显示出正向连接。 3C 分析还表明，BIME 之间的一些联系不依赖于 HU 和 naRNA4；其他 RNA 或蛋白质可能参与建立其他不依赖于 HU 或 naRNA4 的染色体内连接（Qian et al. 2015）。这种非编码 RNA 介导的 DNA 压缩与 ncRNA 在真核生物异染色质基因抑制中的作用相比较（Jorgensen 1990；Yang 和 Li 2016）。
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