Takada Hiraku, Shimada Tomohiro, Dey Debashish, Quyyum M Zuhaib, Nakano Masahiro, Ishiguro Akira, Yoshida Hideji, Yamamoto Kaneyoshi, Sen Ranjan, Ishihama Akira
Research Center for Micro-Nano Technology, Hosei University, Koganei, Tokyo, Japan.
Laboratory for Chemistry and Life Science, Tokyo Institute of Technology, Nagatsuda, Yokohama, Japan.
PLoS One. 2016 Dec 22;11(12):e0163057. doi: 10.1371/journal.pone.0163057. eCollection 2016.
Escherichia coli contains seven rRNA operons, each consisting of the genes for three rRNAs (16S, 23S and 5S rRNA in this order) and one or two tRNA genes in the spacer between 16S and 23S rRNA genes and one or two tRNA genes in the 3' proximal region. All of these rRNA and tRNA genes are transcribed from two promoters, P1 and P2, into single large precursors that are afterward processed to individual rRNAs and tRNAs by a set of RNases. In the course of Genomic SELEX screening of promoters recognized by RNA polymerase (RNAP) holoenzyme containing RpoD sigma, a strong binding site was identified within 16S rRNA gene in each of all seven rRNA operons. The binding in vitro of RNAP RpoD holoenzyme to an internal promoter, referred to the promoter of riRNA (an internal RNA of the rRNA operon), within each 16S rRNA gene was confirmed by gel shift assay and AFM observation. Using this riRNA promoter within the rrnD operon as a representative, transcription in vitro was detected with use of the purified RpoD holoenzyme, confirming the presence of a constitutive promoter in this region. LacZ reporter assay indicated that this riRNA promoter is functional in vivo. The location of riRNA promoter in vivo as identified using a set of reporter plasmids agrees well with that identified in vitro. Based on transcription profile in vitro and Northern blot analysis in vivo, the majority of transcript initiated from this riRNA promoter was estimated to terminate near the beginning of 23S rRNA gene, indicating that riRNA leads to produce the spacer-coded tRNA. Under starved conditions, transcription of the rRNA operon is markedly repressed to reduce the intracellular level of ribosomes, but the levels of both riRNA and its processed tRNAGlu stayed unaffected, implying that riRNA plays a role in the continued steady-state synthesis of tRNAs from the spacers of rRNA operons. We then propose that the tRNA genes organized within the spacers of rRNA-tRNA composite operons are expressed independent of rRNA synthesis under specific conditions where further synthesis of ribosomes is not needed.
大肠杆菌含有七个rRNA操纵子，每个操纵子由三个rRNA基因（依次为16S、23S和5S rRNA）以及16S和23S rRNA基因间隔区中的一两个tRNA基因和3'近端区域中的一两个tRNA基因组成。所有这些rRNA和tRNA基因都从两个启动子P1和P2转录成单一的大前体，随后通过一组核糖核酸酶加工成单个的rRNA和tRNA。在对含有RpoD σ因子的RNA聚合酶（RNAP）全酶识别的启动子进行基因组SELEX筛选过程中，在所有七个rRNA操纵子的每个16S rRNA基因内都鉴定到一个强结合位点。通过凝胶迁移试验和原子力显微镜观察证实，RNAP RpoD全酶在体外与每个16S rRNA基因内的一个内部启动子（称为riRNA启动子，rRNA操纵子的一种内部RNA）结合。以rrnD操纵子内的这个riRNA启动子为代表，使用纯化的RpoD全酶检测到了体外转录，证实该区域存在一个组成型启动子。LacZ报告基因检测表明，这个riRNA启动子在体内具有功能。使用一组报告质粒确定的体内riRNA启动子位置与体外确定的位置非常吻合。基于体外转录谱和体内Northern印迹分析，估计从这个riRNA启动子起始的大部分转录本在23S rRNA基因起始附近终止，这表明riRNA导致产生间隔区编码的tRNA。在饥饿条件下，rRNA操纵子的转录受到显著抑制，以降低细胞内核糖体水平，但riRNA及其加工后的tRNAGlu水平均不受影响，这意味着riRNA在从rRNA操纵子间隔区持续稳定合成tRNA中发挥作用。然后我们提出，在不需要进一步合成核糖体的特定条件下，rRNA - tRNA复合操纵子间隔区内组织的tRNA基因独立于rRNA合成进行表达。
