Supplementary Material for: Investigating the Trans Effects of IS1 Transposases on Intragenomic DNA Movements in Escherichia coli

Introduction: The transposase protein, InsAB′, is the key cytoplastic determinant that catalyzes IS1 movement within a single DNA molecule or between two DNA molecules. Regulation of InsAB′ synthesis, its structural characteristics, and its potent cis effect on IS1 transposition have been subjects of thorough investigation. However, limited efforts have been devoted to measuring the levels of IS1 transcripts and transposases made by native IS1 elements and examining their trans-acting function, particularly concerning their role in transposing otherwise immobile IS1 elements and other target DNA segments within the bacterial genome. Methods: All constructs were made on the E. coli chromosome using the lambda-Red system. IS1 transposition frequencies were determined using Bgl+ mutation assays, colony PCR and DNA sequencing. IS1 transcripts and transposases were quantitated using lacZ transcriptional and translational reporters. Results: We first confirm that a native IS1 element, IS1E, can transpose at a dramatically elevated rate in the absence of both transcriptional and post-transcriptional regulations. Using lacZ reporters targeting the transposase gene insAB′, we reveal that the InsA repressor moderately reduces insAB′ transcription by as much as 16.8 fold. However, the combined action of InsA and ribosomal frameshifting leads to a much stronger reduction in InsAB′ production, by up to 735 fold. We show that only a small percentage (roughly 1.6%) of IS1 transcripts are successfully translated into InsAB′, highlighting the primary role of post-transcriptional regulation over transcriptional repression in governing IS1 transposition. We also demonstrate, for the first time, that high levels of InsAB′ exhibit a strong trans effect, being capable of efficiently transposing not only normally non-mobile IS1 elements but also a larger miniIS1 cassette within the genome, with greater amounts of InsAB′ leading to higher frequencies of transposition. Conclusions: By dissecting and quantitating the roles of transcriptional and translational controls in transposase production, our study reveals that post-transcriptional regulation via ribosomal frameshifting is the central mechanism governing IS1 intragenomic transposition. Our findings demonstrate that IS1 transposases function in trans, mediating the movement of DNA segments specifically bordered by its IRL and IRR sequences. These findings advance our comprehension of how IS elements mobilize within bacterial genomes.

引言：转座酶蛋白InsAB′是催化插入序列1（IS1）在单个DNA分子内部或两个DNA分子之间移动的关键细胞质决定因子。针对InsAB′的合成调控、结构特征及其对IS1转座的强力顺式作用的研究已较为充分。然而，目前针对天然IS1元件产生的IS1转录本与转座酶水平的检测，以及对其反式作用功能的研究仍较为有限，尤其是其在介导原本无法移动的IS1元件及细菌基因组内其他靶DNA片段转座中的相关研究尚少。
方法：本研究所有构建体均通过λ-Red重组系统在大肠杆菌（E. coli）染色体上构建完成。IS1转座频率通过Bgl+突变检测、菌落PCR及DNA测序进行测定。IS1转录本与转座酶的定量分析采用lacZ转录与翻译报告基因系统。
结果：本研究首先证实，天然IS1元件IS1E在同时缺失转录与转录后调控的情况下，转座率可显著升高。通过靶向转座酶基因insAB′的lacZ报告基因，我们发现InsA阻遏蛋白可将insAB′的转录水平适度下调至多16.8倍。然而，InsA与核糖体移码的联合作用可使InsAB′的产生量大幅降低，降幅最高可达735倍。研究显示，仅约1.6%的IS1转录本可成功翻译为InsAB′，这表明在调控IS1转座的过程中，转录后调控相较于转录抑制发挥着主导作用。本研究还首次证实，高浓度的InsAB′具有显著的反式作用效果，可高效介导原本无法移动的IS1元件以及基因组内更大的miniIS1盒式结构的转座，且InsAB′的表达量越高，转座频率也越高。
结论：通过解析并定量分析转录与翻译调控在转座酶产生过程中的作用，本研究揭示，基于核糖体移码的转录后调控是调控IS1基因组内转座的核心机制。本研究结果证实，IS1转座酶可通过反式作用介导以其反向末端重复序列IRL与IRR为边界的DNA片段的移动。这些发现加深了我们对插入序列在细菌基因组内移动机制的理解。