Genetic Organization Analysis of the Major Cold-Shock Gene deaD in Escherichia coli

DeaD or CsdA (cold-shock DEAD box protein A), is an ATP-dependent RNA helicase, a major cold-shock protein that plays key roles in translation initiation, ribosome biogenesis, and mRNA decay at low temperatures in bacteria. The DeaD homolog of Escherichia coli is ubiquitously present in eukaryotes, archaea, and bacteria. DeaD has been extensively studied at the protein level in E. coli. However, the complex mechanism of deaD gene regulation is yet to be deciphered. To study deaD gene regulation, we engineered a promoter-less reporter plasmid vector which contains an ORF of green fluorescence protein (GFP) without a promoter region. We performed sequential, incremental, zone-wise cloning of DNA fragments of the upstream region of the deaD ORF and analyzed GFP expression in our promoter-less plasmid vector to identify the promoter region. We found out the promoter around 800 nucleotides upstream of deaD ORF, which was further confirmed by its In Vivo deletion in the E. coli genome. We observed the expression of the deaD gene might also occur from the immediate upstream of the nlpI and pnp gene, revealing the phenomenon of an operon system. Interestingly, we found the short ORF of the gene yrbN overlaps with the ORF of deaD but not in the frame. Subsequently, Multiple Sequence Alignment profiles showed that not only the promoter and the unusually long 5'UTR region but the whole genetic arrangement of the deaD gene, including the overlapping phenomenon of ORF of the yrbN gene, is conserved in Gamma-proteobacteria indicating a conserved gene expression pattern.