Question

A scientist wishes to express a eukaryotic protein in bacterial cells. The gene is cloned along with its promoter region and is inserted into a plasmid. After transforming the plasmid into bacterial cells, protein expression is initiated, but no protein is observed after the cells are lysed. Why? How could this problem be fixed?

Answer 1

promoter is a regulatory region of DNA located upstream (towards the 5' region) of of a gene, providing a control point for regulated gene transcription. The promoter contains specific DNA sequences that are recognized by proteins known as transcription factors. These factors bind to the promoter sequences, recruiting RNA polymerase.

Promoter regions are different from prokaryotes to eukarotes.

In  prokaryotes, the promoter consists of two short sequences at -10 and -35 positions upstream from the transcription start site. The sequence at -10 is called the Pribnow box, or the -10 element, and usually consists of the six nucleotides TATAAT. The Pribnow box is absolutely essential to start transcription in prokaryotes. The other sequence at -35 (the -35 element) usually consists of the six nucleotides TTGACA. Its presence allows a very high transcription rate.

Eukaryotic promoters are typically lie upstream of the gene and can have regulatory elements several kilobases away from the transcriptional start site. In eukaryotes, the transcriptional complex can cause the DNA to bend back on itself, which allows for placement of regulatory sequences far from the actual site of transcription. Many eukaryotic promoters, contain a TATA box (sequence TATAAA), which in turn binds a TATA binding protein which assists in the formation of the RNA polymerase transcriptional complex. The TATA box typically lies very close to the transcriptional start site (often within 50 bases).

Thus, an eukaryotic promoter doesn't allow the gene to be transcribed in a prokaryotic cell. So if a gene with has to be transcribed, it's eukaryotic promoter must be removed and prokaryotic promoter has to be ligated and then the transformation has to be conducted, which then allows the eukaryotic gene gets transcribed in prokaryotes.