Sequence and thermodynamic characteristics of terminators revealed by FlowSeq and the discrimination of terminators strength

Zhai, Weiji; Duan, Yanran; Zhang, Xiaomei; Xu, Guoqiang; Li, Hui; Shi, Jin‐Song; Xu, Zhenghong; Zhang, Xiaojuan

doi:10.1016/j.synbio.2022.06.003

Cited by 4 publications

(4 citation statements)

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“…Application of the Flow-seq method is not limited to the provided examples. This technique is also employed to evaluate (using reporter constructs as biosensors in various bacterial strains, including knockout ones [ 92 ]) the effects on the glycolytic processes, assess terminator sequences [ 93 ], identify the genes involved in the changes in a particular metabolic pathway (using biosensor constructs [ 94 ]), and solve other problems (e.g., study splicing) [ 95 ].…”

Section: The Scheme Of the Flow-seq Technique The Features And Result...mentioning

confidence: 99%

“…Применение метода Flow-seq не ограничивается приведенными примерами. Этот метод применяют также для оценки (с помощью репортерных конструкций как биосенсоров в различных штаммах бактерий, в том числе и нокаутных [92]) эффектов, влияющих на гликолитические процессы, для оценки терминаторных последовательностей [93], определения генов, вовлекаемых в изменения конкретного метаболического пути (с использованием биосенсорных конструкций [94]) и решения других задач, например, исследования сплайсинга [95].…”

Section: схема метода Flow-seq особенности и результаты его примененияunclassified

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Flow-Seq Method: Features and Application in Bacterial Translation Studies

et al. 2023

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The Flow-seq method is based on using reporter construct libraries, where a certain element regulating the gene expression of fluorescent reporter proteins is represented in many thousands of variants. Reporter construct libraries are introduced into cells, sorted according to their fluorescence level, and then subjected to next-generation sequencing. Therefore, it turns out to be possible to identify patterns that determine the expression efficiency, based on tens and hundreds of thousands of reporter constructs in one experiment. This method has become common in evaluating the efficiency of protein synthesis simultaneously by multiple mRNA variants. However, its potential is not confined to this area. In the presented review, a comparative analysis of the Flow-seq method and other alternative approaches used for translation efficiency evaluation of mRNA was carried out; the features of its application and the results obtained by Flow-seq were also considered.

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Section: The Scheme Of the Flow-seq Technique The Features And Result...mentioning

confidence: 99%

Section: схема метода Flow-seq особенности и результаты его примененияunclassified

Flow-Seq Method: Features and Application in Bacterial Translation Studies

et al. 2023

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“…The notion that VPA1349 T with a computed Δ G T value of −8.80 kcal/mol is a less stable terminator is consistent with the results previously obtained by Zhai, et al who classified the 214 characterized terminators in E . coli into strong and weak terminators, with the strong terminators having a low Δ G T of −17 to −14 kcal/mol [ 50 ]. A strong and stable hairpin is expected to cause efficient termination due to a greater ability to push the RNAP away from the DNA strand [ 51 ].…”

Section: Discussionmentioning

confidence: 99%

“…nd, not detected; *, p < 0.05, as revealed by one-way ANOVA followed by Dunnett's multiple comparison test. (C) Schematic structure of the general intrinsic terminator (adapted from [50] with modifications). (TIF) S4 Fig.…”

Section: Discussionmentioning

confidence: 99%

The read-through transcription-mediated autoactivation circuit for virulence regulator expression drives robust type III secretion system 2 expression in Vibrio parahaemolyticus

Anggramukti,

Ishii,

Pratama

et al. 2024

PLoS Pathog

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Vibrio parahaemolyticus is the leading cause of seafood-borne gastroenteritis in humans worldwide. The major virulence factor responsible for the enteropathogenicity of this pathogen is type III secretion system 2 (T3SS2), which is encoded on the 80-kb V. parahaemolyticus pathogenicity island (Vp-PAI), the gene expression of which is governed by the OmpR-family transcriptional regulator VtrB. Here, we found a positive autoregulatory feature of vtrB transcription, which is often observed with transcriptional regulators of bacteria, but the regulation was not canonically dependent on its own promoter. Instead, this autoactivation was induced by heterogeneous transcripts derived from the VtrB-regulated operon upstream of vtrB. VtrB-activated transcription overcame the intrinsic terminator downstream of the operon, resulting in transcription read-through with read-in transcription of the vtrB gene and thus completing the autoregulatory loop for vtrB gene expression. The dampening of read-through transcription with an exogenous strong terminator reduced vtrB gene expression. Furthermore, a V. parahaemolyticus mutant with defects in the vtrB autoregulatory loop also showed compromises in T3SS2 expression and T3SS2-dependent cytotoxicity in vitro and enterotoxicity in vivo, indicating that this autoregulatory loop is essential for sustained vtrB activation and the consequent robust expression of T3SS2 genes for pathogenicity. Taken together, these findings demonstrate that the regulatory loop for vtrB gene expression based on read-through transcription from the upstream operon is a crucial pathway in T3SS2 gene regulatory network to ensure T3SS2-mediated virulence of V. parahaemolyticus.

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