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The Effect of Translation Promoting Site (TPS) on Protein Expression in E. coli Cells

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Abstract

The study of translation initiation in prokaryotes assumes that there should be a mechanism different from the canonical model, which postulates the formation of the pre-initiation complex through the interaction of the Shine-Dalgarno sequence (SD) at the 5′-end of mRNA and the anti-Shine-Dalgarno site at the 3′-end of 16S rRNA. In this paper we’ve studied the effect of TPS (Translation-initiation Promoting Site) on β-glucuronidase expression in E. coli cells at different cultivation temperatures. The examined leader sequences were cloned into the pET23c plasmid upstream the β-glucuronidase gene; protein expression was performed in E. coli BL21 (DE3) cells. β-glucuronidase activity was measured in bacterial cell extracts via paranitrophenyl b-d-glucuronide assay. The quantity of expressed protein was measured by Western blotting with following densitometry. It was shown that TPS increases the level of protein expression at stressful conditions (10 °C and 44 °C) 5–8 times compared to control. The combination of TPS and SD sites in the 5′-leader sequence of the mRNA created an enhancer that increased the expression level 2–3.6 times compared to a single SD-sequence. Based on the obtained data and the computer modeling of interaction between 16S rRNA and TPS, we proposed an alternative variation of prokaryotic translation initiation.

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Acknowledgements

This study was supported by the Kazakhstan Science Committee grants with the Project Numbers AP05130800 and AP05132532.

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Authors and Affiliations

  1. Laboratory of Protein and Nucleic Acids, M.Aythozhin Institute of Molecular Biology and Biochemistry, Dosmuhamedov St. 80, 050012, Almaty, Kazakhstan

    Anna Nizkorodova, Andrey Zhigailov & Bulat Iskakov

  2. Department of Biodiversity, Biological Faculty, Al-Faraby State National University, Almaty, Kazakhstan

    Maria Suvorova

Authors
  1. Anna Nizkorodova
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  2. Maria Suvorova
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  3. Andrey Zhigailov
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  4. Bulat Iskakov
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Contributions

AN and BI conceived and designed the experiments. AN, MS and AZ performed the experiments. AN analyzed and/or interpreted the data. AN wrote the manuscript.

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Correspondence to Anna Nizkorodova.

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The authors Nizkorodova A., Suvorova M., Zhigailov A., and Iskakov B. declare that they have no conflicts of interest related to the subject matter or materials discussed in this article.

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Supplementary file1 (TIF 1341 kb)

Fig. S1 β-glucuronidase activity at temperature stress condition relative to that at 37°C. GUS-activity in cells cultivated at 37°C was accepted as 100% for each construction; the relative GUS-activity values for stress temperatures are shown. The asterisk indicates the data sets significantly (p ≤ 0.05) differing from those at 37°C for each of the constructions.

Supplementary file2 (TIF 1191 kb)

Fig. S2 Graphical representation of suggested and canonical models of prokaryotic translation initiation. Standard conformation of small ribosomal subunit 30S is shown in beige color and conformation that appears under the cold shock condition – in purple color (with changed position of anti-Shine-Dalgarno sequence).

Supplementary file3 (TIF 765 kb)

Fig. S3 Secondary structure of leaders ‘TPSfar’ (a), ‘TPS__SD’ (b), ‘TPSlongSD’ (c). Start codon is shown in red, TPS – in blue, SD – in green.

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Nizkorodova, A., Suvorova, M., Zhigailov, A. et al. The Effect of Translation Promoting Site (TPS) on Protein Expression in E. coli Cells. Mol Biotechnol 62, 326–334 (2020). https://doi.org/10.1007/s12033-020-00251-1

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