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.
Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
