Unprecedented high-resolution view of bacterial operon architecture revealed by RNA sequencing.
Unprecedented high-resolution view of bacterial operon architecture revealed by RNA sequencing.
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DOI：
10.1128/mbio.01442-14
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发表时间：
2014-07-08
期刊：
影响因子：
6.4
通讯作者：
Wanner BL
中科院分区：
文献类型：
--
作者：
Conway T;Creecy JP;Maddox SM;Grissom JE;Conkle TL;Shadid TM;Teramoto J;San Miguel P;Shimada T;Ishihama A;Mori H;Wanner BL
我们分析了转录组的大肠杆菌K-12的链特异性RNA测序在稳定状态（代谢期）的增长过程中，在葡萄糖基本培养基中进入稳定期的单核苷酸分辨率。为了生成高分辨率的转录组图谱，我们开发了一种组织模式，该模式表明在实践中仅需要三个特征来定义操纵子架构：启动子、终止子和深度RNA序列读段覆盖率。我们精确地注释了2，122个启动子和1，774个终止子，定义了1，510个操纵子，平均每个操纵子包含1.98个基因。我们的分析揭示了大肠杆菌操纵子结构的前所未有的观点。大部分（36%）的操纵子是复杂的内部启动子或终止子，产生多个转录单位。对于43%的操纵子，我们观察到多顺反子基因的差异表达，尽管在相同的操纵子中，表明大肠杆菌操纵子结构允许基因表达的微调。我们发现370个会聚操纵子中有276个无效终止，产生平均重叠286个核苷酸的互补3′转录物末端，388个发散操纵子中有136个具有启动子排列，使得它们的5′末端平均重叠168个核苷酸。我们发现了89个反义转录的397个核苷酸的平均长度，7个未注释的转录内的基因间区域，和18个正义转录完全重叠的操纵子上的相对链。在519个重叠转录物中，75%对应于大肠杆菌中高度保守的序列（>50个基因组）。我们的数据扩展了最近的研究，显示了几种细菌中意想不到的转录组复杂性，并表明反义RNA调控是普遍存在的。我们通过使用单核苷酸分析方法精确定位了大肠杆菌K-12基因组中RNA转录物的5′和3′末端。我们得到的高分辨率转录组图谱显示，ca。三分之一的大肠杆菌操纵子是复杂的，内部启动子和终止子产生多个转录单位，并允许这些操纵子内的差异基因表达。我们发现了广泛的反义转录，结果从500多个操纵子，完全重叠或广泛重叠相邻的发散或收敛操纵子。对应于这些反义转录物的基因组区域在大肠杆菌（包括志贺氏菌属物种）中高度保守，尽管它们是否具有功能仍有待证明。我们对单核苷酸转录组作图发现的特征的观察表明，细菌中更深层的转录调控可能在未来被揭示。
We analyzed the transcriptome of Escherichia coli K-12 by strand-specific RNA sequencing at single-nucleotide resolution during steady-state (logarithmic-phase) growth and upon entry into stationary phase in glucose minimal medium. To generate high-resolution transcriptome maps, we developed an organizational schema which showed that in practice only three features are required to define operon architecture: the promoter, terminator, and deep RNA sequence read coverage. We precisely annotated 2,122 promoters and 1,774 terminators, defining 1,510 operons with an average of 1.98 genes per operon. Our analyses revealed an unprecedented view of E. coli operon architecture. A large proportion (36%) of operons are complex with internal promoters or terminators that generate multiple transcription units. For 43% of operons, we observed differential expression of polycistronic genes, despite being in the same operons, indicating that E. coli operon architecture allows fine-tuning of gene expression. We found that 276 of 370 convergent operons terminate inefficiently, generating complementary 3′ transcript ends which overlap on average by 286 nucleotides, and 136 of 388 divergent operons have promoters arranged such that their 5′ ends overlap on average by 168 nucleotides. We found 89 antisense transcripts of 397-nucleotide average length, 7 unannotated transcripts within intergenic regions, and 18 sense transcripts that completely overlap operons on the opposite strand. Of 519 overlapping transcripts, 75% correspond to sequences that are highly conserved in E. coli (>50 genomes). Our data extend recent studies showing unexpected transcriptome complexity in several bacteria and suggest that antisense RNA regulation is widespread. We precisely mapped the 5′ and 3′ ends of RNA transcripts across the E. coli K-12 genome by using a single-nucleotide analytical approach. Our resulting high-resolution transcriptome maps show that ca. one-third of E. coli operons are complex, with internal promoters and terminators generating multiple transcription units and allowing differential gene expression within these operons. We discovered extensive antisense transcription that results from more than 500 operons, which fully overlap or extensively overlap adjacent divergent or convergent operons. The genomic regions corresponding to these antisense transcripts are highly conserved in E. coli (including Shigella species), although it remains to be proven whether or not they are functional. Our observations of features unearthed by single-nucleotide transcriptome mapping suggest that deeper layers of transcriptional regulation in bacteria are likely to be revealed in the future.