Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX 78712, USA; Department of Molecular Biosciences, University of Texas at Austin, Austin, TX 78712, USA.
Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX 78712, USA; Department of Molecular Biosciences, University of Texas at Austin, Austin, TX 78712, USA.
J Mol Biol. 2018 Aug 17;430(17):2760-2783. doi: 10.1016/j.jmb.2018.06.019. Epub 2018 Jun 15.
The thermostable Geobacillus stearothermophilus GsI-IIC intron is among the few bacterial group II introns found to proliferate to high copy number in its host genome. Here, we developed a bacterial genetic assay for retrohoming and biochemical assays for protein-dependent and self-splicing of GsI-IIC. We found that GsI-IIC, like other group IIC introns, retrohomes into sites having a 5'-exon DNA hairpin, typically from a bacterial transcription terminator, followed by short intron-binding sequences (IBSs) recognized by base pairing of exon-binding sequences (EBSs) in the intron RNA. Intron RNA insertion occurs preferentially but not exclusively into the parental lagging strand at DNA replication forks, using a nascent lagging strand DNA as a primer for reverse transcription. In vivo mobility assays, selections, and mutagenesis indicated that a variety of GC-rich DNA hairpins of 7-19 bp with continuous base pairs or internal elbow regions support efficient intron mobility and identified a critically recognized nucleotide (T-5) between the hairpin and IBS1, a feature not reported previously for group IIC introns. Neither the hairpin nor T-5 is required for intron excision or lariat formation during RNA splicing, but the 5'-exon sequence can affect the efficiency of exon ligation. Structural modeling suggests that the 5'-exon DNA hairpin and T-5 bind to the thumb and DNA-binding domains of GsI-IIC reverse transcriptase. This mode of DNA target site recognition enables the intron to proliferate to high copy number by recognizing numerous transcription terminators and then finding the best match for the EBS/IBS interactions within a short distance downstream.
嗜热芽胞杆菌 GsI-IIC 内含子是少数在宿主基因组中大量增殖的细菌Ⅱ类内含子之一。在这里，我们开发了细菌遗传反式剪接测定法和蛋白质依赖的生化测定法以及 GsI-IIC 的自我剪接测定法。我们发现，与其他 I 类内含子一样，GsI-IIC 反式剪接进入具有 5'-exon DNA 发夹结构的位点，通常来自细菌转录终止子，然后是短的内含子结合序列（IBS），这些序列通过内含子 RNA 中的exon 结合序列（EBS）的碱基配对被识别。内含子 RNA 的插入优先但不是排他地发生在 DNA 复制叉处的亲本滞后链上，使用新生的滞后链 DNA 作为反转录的引物。体内迁移测定、选择和突变分析表明，7-19 个碱基对的富含 GC 的连续碱基对或内部肘区的 DNA 发夹结构支持有效的内含子迁移，并鉴定了发夹和 IBS1 之间关键识别核苷酸（T-5），这一特征以前未在 I 类内含子中报道过。无论是发夹还是 T-5，对于 RNA 剪接过程中的内含子切除或套索形成都不是必需的，但 5'-exon 序列可以影响外显子连接的效率。结构建模表明，5'-exon DNA 发夹和 T-5 结合到 GsI-IIC 逆转录酶的拇指和 DNA 结合结构域。这种 DNA 靶位点识别模式使内含子能够通过识别众多转录终止子来大量增殖，然后在短距离下游找到 EBS/IBS 相互作用的最佳匹配。
