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During gene transcription, RNA polymerase initiates the process by recognizing the promoter sequence, and terminates it upon recognizing the terminator sequence located at the 3′-UTR, leading to dissociation of the transcription complex. Therefore, promoters and terminators within the transcription unit play the role of initiating and terminating transcription, respectively. For downstream transcription units, in addition to the direct effect of terminating transcript read-through, the dissociation of the RNA polymerase from the terminator may affect the binding of the promoter to RNA polymerase in the subsequent transcription unit, thus indirectly altering the expression of the downstream transcription unit. This interplay between terminators and promoters across transcription units remains poorly understood, therefore, elucidating the impact of terminators on the transcriptional strength of downstream transcription units is of great significance for the precise regulation of gene expression and the development of efficient terminators. In this study, a library containing 405 different combinatorial elements (terminator-spacer-promoter) was constructed by combining nine terminators, five spacer sequences, and nine promoters using one-pot assembly technology. All combinations in the library were sequenced and analyzed in terms of fluorescence intensity based on the FlowSeq technology to establish the correlations between combinatorial sequences and downstream gene expression. The results showed that combinations of weak terminators, short spacers, and strong terminators were more favorable to enhance the expression of downstream genes, while combinations of strong terminators, long spacers, and weak terminators reduced the expression of downstream genes. Quantitative analysis of transcription revealed that weak terminators not only enhanced downstream leakage transcription (21~70-fold enhancement), but also facilitated downstream promoters to re-recruit RNA polymerase for re-promoted transcription (2~3-fold enhancement). This study has elucidated the effect and mechanism of terminators on the regulation of gene expression in the downstream transcription units, providing a design framework for the construction of gene circuits using terminators.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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