Rajani MallaParajuli, Damodar2026-04-272026-04-272015https://hdl.handle.net/20.500.14540/264605-Aminolevulinic acid (ALA) is the main precursor for tetrapyrrole containing biological molecules that include chlorophyll, cytochrome C for respiration, cytochrome P450 for drug metabolism, seroheme for cysteine biosynthesis, corrin ring of vitamin B12 molecule, having implication in diverse biological system from eubacteria to plants and animals including mammals. One of the limitations in the pharmaceutical or chemical industries is to study the drug metabolism profiles of new pharmaceutical entities or new chemical compound that requires either rat liver microsome, S9 mix of phenobarbital (carcinogen) induced liver enzyme system, and eventually human liver microsomes. This system has been studied for the metabolism of carcinogens and their genotoxicity in bacterial system. However, in these bacterial expression systems the expression amount is compromised as it requires expensive ALA as the precursor. However due to tight regulation by different mechanisms with or without involving enzymes, these critically important molecules are very rarely synthesized by Microorganisms. Many works have been done to overexpress gene biosynthesizing ALA for expression of cytochrome P450 isoforms. There might also be RNA involved regulation in ALA biosynthetic genes and subsequent stages of heme biosynthesis by Riboswitch like mechanism. Here if new regulatory mechanism is stipulated the protein and chromosomal engineering will be designed for sequence playing role in Riboswitch like mechanism to mimic this effect. However, very less of the works are involved that has looked in to the untranslated RNA region and role of their folding structure that would reduce amount of ALA and final product. Here mfold structure is developed for 5’UTR of heme biosynthetic genes and their thermal stability and GC content is analysed. We selected upstream UTR of 10 heme biosynthetic genes as mentioned in ecocys. Out of 10 genes transcript detail of only five genes were available and for rest of five genes upstream 100 nucleotide from translation initiation site were selected for structural study. In heme biosynthetic pathway hemN and hemH showed folding structure that could regulate heme biosynthesis by Riboswitch like mechanism. Similarly UTR of hemB gene also showed more stable structure than that of other with length of nucleotide and GC percentage comparable. There was no sequence similarity of UTR of all heme Biosynthetic genes in Escherichia coli when studied through BLAST (alignment tool). We designed PCR amplification primers of hemA gene of E coli. After PCR amplification heme gene is also been successfully cloned in pET15b expression vector and pUC19. Key words: heme, hemA, UTR, riboswitchen-USRiboswitchUTRThesis