Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Institute of Biotechnology, Shanxi Universitygrid.163032.5, Taiyuan, Shanxi Province, China.
Institute of Applied Chemistry, Shanxi Universitygrid.163032.5, Taiyuan, Shanxi Province, China.
Microbiol Spectr. 2022 Apr 27;10(2):e0165621. doi: 10.1128/spectrum.01656-21. Epub 2022 Mar 14.
The extracellular substrate-binding proteins (SBPs) of ATP-binding cassette (ABC) importers tend to be expressed in excess relative to their cognate translocators, but how the stoichiometry of ABC transporters is controlled remains unclear. Here, we elucidated a mechanism contributing to differential gene expression in operons encoding ABC importers by employing cellulolytic Clostridia species, specifically Ruminiclostridium cellulolyticum. We found that there were usually stem-loop structures downstream of SBP genes, which could prematurely terminate the transcription of ABC importers and were putative internal intrinsic terminators, resulting in high transcript levels of upstream SBP genes and low transcript levels of downstream cognate translocator genes. This was determined by their termination efficiencies. Internal terminators had a lower U content in their 3' U-rich tracts and longer GC-rich stems, which distinguishes them from canonical terminators and potentially endows them with special termination efficiencies. The pairing of U-rich tracts and the formation of unpaired regions in these internal terminators contributed to their folding energies, affecting the stability of their upstream SBP transcripts. Our findings revealed a strategy of internal transcriptional terminators controlling stoichiometry of their flanking transcripts. Operons encoding protein complexes or metabolic pathways usually require fine-tuned gene expression ratios to create and maintain the appropriate stoichiometry for biological functions. In this study, a strategy for controlling differential expression of genes in an operon was proposed by utilizing ABC importers from Ruminiclostridium cellulolyticum. We found that a stem-loop structure is introduced into the intergenic regions of operons encoding ABC importers as the putative internal terminator, which results in the premature termination of transcription. Consequently, the stoichiometric ratio of genes flanking terminators is precisely determined by their termination efficiencies and folding energies at the transcriptional level. Thus, it can be utilized as a promising synthetic biology tool to control the differential expression of genes in an operon.
ABC 转运蛋白的细胞外基质结合蛋白 (SBPs) 相对于其同源转运蛋白往往表达过量，但 ABC 转运蛋白的化学计量如何控制仍不清楚。在这里，我们通过使用纤维素分解梭菌物种，特别是 Ruminiclostridium cellulolyticum，阐明了导致编码 ABC 进口器操纵子中差异基因表达的机制。我们发现 SBP 基因的下游通常存在茎环结构，这些结构可以使 ABC 进口器的转录提前终止，并且是潜在的内部内在终止子，导致上游 SBP 基因的转录物水平较高，而下游同源转运蛋白基因的转录物水平较低。这是通过它们的终止效率来确定的。内部终止子在其 3' U 丰富的片段中具有较低的 U 含量和较长的 GC 丰富的茎，这将它们与典型的终止子区分开来，并可能赋予它们特殊的终止效率。富含 U 的片段的配对以及这些内部终止子中未配对区域的形成有助于它们的折叠能量，从而影响它们上游 SBP 转录物的稳定性。我们的发现揭示了内部转录终止子控制其侧翼转录物化学计量的策略。编码蛋白质复合物或代谢途径的操纵子通常需要精细调节基因表达比，以产生和维持适当的化学计量比以实现生物学功能。在这项研究中，提出了一种通过利用 Ruminiclostridium cellulolyticum 中的 ABC 进口器来控制操纵子中基因差异表达的策略。我们发现，茎环结构被引入到编码 ABC 进口器的操纵子的基因间区域作为潜在的内部终止子，导致转录提前终止。因此，终止子侧翼基因的化学计量比在转录水平上通过其终止效率和折叠能量精确确定。因此，它可以用作一种有前途的合成生物学工具，用于控制操纵子中基因的差异表达。
