Towards identifying the promoter site of csdA gene of Escherichia coli

Ajayanath, E. (2019) Towards identifying the promoter site of csdA gene of Escherichia coli. Masters thesis, Indian Institute of Science Education and Research Kolkata.

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Abstract

Micro-organisms have to adapt to various challenging environments including osmotic stress, nutrient, and oxygen availability and fluctuations in temperature. After temperature decrease, some key changes occur in cell physiology and metabolism, like changes in the membrane fluidity and changes in nucleic acid secondary structures. All these deleterious effects are counteracted by induction of specific proteins known as Cold Shock Proteins (CSPs). Major cold shock protein first identified was CspA. Eight more proteins which are identified to be homologous to the CspA protein are found in E.coli; they are named in alphabetical order from CspB to CspI. CspB, CspG, CspI are induced during the temperature downshift. Proteins that help in restructuring the RNA can be of different types:(1)RNA chaperones which disintegrate the misfolded RNA helping in the production of suitably folded RNA,(2)RNA helicases,(3)RNA annealers which speed up the annealing of complementary RNAs,(4)RNA binding proteins helping in stabilizing their specific structures. CspA protein comes under the first category of proteins having chaperone activity, but CsdA is a member of the DEAD box family of ATP-dependent RNA helicases which helps in RNA splicing, ribosome biogenesis, translational initiation, and mRNA degradation. In bacteria, 5´UTR regions of these CSPs are unusually long and regulates the stability of its mRNA and their protein production under cold acclimation phase. CsdA mRNA primer extension studies conducted by Toone et al., 1991 reported an extended 5´UTR region of 226 base pair in length and predicted a possible promoter site directly upstream of that region. Experiments done by cloning the suspected promoter site into a GFP protein expressing vector indicated an absence of promoter site upstream of 226 base pair 5´UTR region. Our studies put forward a promoter site 800 base pair upstream of the csdA gene. We validated these studies by quantitative assessment of mRNA production by the cloned promoter sites.

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