Engineering of <i>Bacillus</i> Promoters Based on Interacting Motifs between UP Elements and RNA Polymerase (RNAP) α-Subunit
<i>Bacillus</i> genetics need more versatile promoters for gene circuit engineering. UP elements are widely distributed in noncoding regions and interact with the α-subunit of RNA polymerase (RNAP). They can be applied as a standard element for synthetic biology. Characterization of the...
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| Format: | Article |
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MDPI AG
2022-11-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/23/21/13480 |
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| author | Youran Li Xufan Ma Liang Zhang Zhongyang Ding Sha Xu Zhenghua Gu Guiyang Shi |
| author_facet | Youran Li Xufan Ma Liang Zhang Zhongyang Ding Sha Xu Zhenghua Gu Guiyang Shi |
| author_sort | Youran Li |
| collection | DOAJ |
| description | <i>Bacillus</i> genetics need more versatile promoters for gene circuit engineering. UP elements are widely distributed in noncoding regions and interact with the α-subunit of RNA polymerase (RNAP). They can be applied as a standard element for synthetic biology. Characterization of the binding motif between UP elements and RNAP may assist with rational and effective engineering. In this study, 11 <i>Bacillus</i> constitutive promoters were screened for strength in <i>Bacillus licheniformis</i>. The motif in UP elements from a strong native promoter, P<sub>Lan</sub>, was characterized. The influence of specific sequences on RNAP binding and expression strength was investigated both in vitro and in vivo. It was found that sequences up to 50 base pairs upstream of the consensus motif significantly contributed to α-CTD (the alpha subunit carboxy-terminal domain) association. Meanwhile, two repeats of a proximal subsite were able to more strongly activate the expression (by 8.2-fold) through strengthening interactions between UP elements and RNAP. Based the above molecular basis, a synthetic UP element, UP5-2P, was constructed and applied to nine wild-type promoters. Fluorescence polarization results demonstrated that it had an apparent effect on promoter–α-CTD interactions, and elevated expression strength was observed for all the engineered promoters. The highest improved core promoter, P<sub>acpp</sub>, was more strongly activated by 7.4-fold. This work thus develops a novel strategy for <i>Bacillus</i> promoter engineering. |
| first_indexed | 2024-03-09T18:59:34Z |
| format | Article |
| id | doaj.art-5c1a3d98dc3b4fc2bf67dba20279eebc |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-09T18:59:34Z |
| publishDate | 2022-11-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | International Journal of Molecular Sciences |
| spelling | doaj.art-5c1a3d98dc3b4fc2bf67dba20279eebc2023-11-24T05:08:39ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672022-11-0123211348010.3390/ijms232113480Engineering of <i>Bacillus</i> Promoters Based on Interacting Motifs between UP Elements and RNA Polymerase (RNAP) α-SubunitYouran Li0Xufan Ma1Liang Zhang2Zhongyang Ding3Sha Xu4Zhenghua Gu5Guiyang Shi6Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, ChinaKey Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, ChinaKey Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, ChinaKey Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, ChinaKey Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, ChinaKey Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, ChinaKey Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China<i>Bacillus</i> genetics need more versatile promoters for gene circuit engineering. UP elements are widely distributed in noncoding regions and interact with the α-subunit of RNA polymerase (RNAP). They can be applied as a standard element for synthetic biology. Characterization of the binding motif between UP elements and RNAP may assist with rational and effective engineering. In this study, 11 <i>Bacillus</i> constitutive promoters were screened for strength in <i>Bacillus licheniformis</i>. The motif in UP elements from a strong native promoter, P<sub>Lan</sub>, was characterized. The influence of specific sequences on RNAP binding and expression strength was investigated both in vitro and in vivo. It was found that sequences up to 50 base pairs upstream of the consensus motif significantly contributed to α-CTD (the alpha subunit carboxy-terminal domain) association. Meanwhile, two repeats of a proximal subsite were able to more strongly activate the expression (by 8.2-fold) through strengthening interactions between UP elements and RNAP. Based the above molecular basis, a synthetic UP element, UP5-2P, was constructed and applied to nine wild-type promoters. Fluorescence polarization results demonstrated that it had an apparent effect on promoter–α-CTD interactions, and elevated expression strength was observed for all the engineered promoters. The highest improved core promoter, P<sub>acpp</sub>, was more strongly activated by 7.4-fold. This work thus develops a novel strategy for <i>Bacillus</i> promoter engineering.https://www.mdpi.com/1422-0067/23/21/13480promoter engineering<i>Bacillus licheniformis</i>UP elementα-CTD |
| spellingShingle | Youran Li Xufan Ma Liang Zhang Zhongyang Ding Sha Xu Zhenghua Gu Guiyang Shi Engineering of <i>Bacillus</i> Promoters Based on Interacting Motifs between UP Elements and RNA Polymerase (RNAP) α-Subunit International Journal of Molecular Sciences promoter engineering <i>Bacillus licheniformis</i> UP element α-CTD |
| title | Engineering of <i>Bacillus</i> Promoters Based on Interacting Motifs between UP Elements and RNA Polymerase (RNAP) α-Subunit |
| title_full | Engineering of <i>Bacillus</i> Promoters Based on Interacting Motifs between UP Elements and RNA Polymerase (RNAP) α-Subunit |
| title_fullStr | Engineering of <i>Bacillus</i> Promoters Based on Interacting Motifs between UP Elements and RNA Polymerase (RNAP) α-Subunit |
| title_full_unstemmed | Engineering of <i>Bacillus</i> Promoters Based on Interacting Motifs between UP Elements and RNA Polymerase (RNAP) α-Subunit |
| title_short | Engineering of <i>Bacillus</i> Promoters Based on Interacting Motifs between UP Elements and RNA Polymerase (RNAP) α-Subunit |
| title_sort | engineering of i bacillus i promoters based on interacting motifs between up elements and rna polymerase rnap α subunit |
| topic | promoter engineering <i>Bacillus licheniformis</i> UP element α-CTD |
| url | https://www.mdpi.com/1422-0067/23/21/13480 |
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