Question

How were core promoter elements identified?

How would you do this in a new organ

Answer 1

Promoters are regulatory sequences in a genome which precede the 5' end of a protein coding region, or exon, and binds to the RNA polymerase enzyme which brings about transcription of the exon downstream of the promoter. A promoter is very essential to be recognized by the RNA polymerase, since prper recognition of promoters leads to the beginning of transcription in the transcription start sites (TSS). The core promoter is made up of promoter elements which include the polymerase binding site, TSS and regulatory stretches of DNA that are essential for establishing trascription. Therefore, the core promoter elements are important components of the cell machinery and therefore needed to be identified and characterized.

In 1966, scientists Scaife and Beckwith hypothesized that it is possible that specific regions of the genome are responsible for carrying out gene expression. They identified the role of mutations in altering the expression of genes, which led to this hypothesis. 1968, Ippen et al. used bacterial strains with mutations at the starting point of the Lac operon and identified a control region which seemed to regulate transcription. They called this control region the promoter and the intiating site of transcription.

In 1979-80, Weil et al. and Manley et al. used in-vitro transcription to identify promoter elements and though this turned out to be a successful attempt in identifying promoter regions, the regulatory roles of a promoter could not be studied in vitro.

Ota et al., also in the late 1970s, came up with the reporter gene assay system, wherein a 'reporter gene' whose expression can be manifested in a visible form, is joined with the promoter. Using this procedure, the levels of promoter activity could be assayed by estimating the expression level of the reporter gene. Since the promoter would be responsible in controlling the transcription and subsequent expression of the reporter gene as well, higher the expression of the reporter, more active the promoter is. This method has been successfully applied using various reporter gene systems including the GFP (green fluorescent protein), luciferase, chloramphenicol acyltransferase etc.

The reporter gene is articially introduced into the cell using a 4-6 kb plasmid which is engineered to code for the reporter gene. The plasmid is also made to code for an antibiotic reporter gene, which is essential for selection of ceels where transcription has taken place succesfully. The plasmid containing the reporter gene and the antibiotic-resistance gene has a multiple cloning site which helps its integration into the chromosomal DNA via homologous recombination upon transfection of the plasmid into the cell. If the promoter is active, it carries out transcription of the reporter gene and the antibiotic resistance gene in the cell. The cells are later treated with the antibiotic whose resistance gene was introduced into the cell. If recombination of the plasmid and transcription by the promoter has been succesful, the cells would survive the antibiotic treatment, while the non-recombinant cells would die. This is how the core promoter region was identified. Further experiments using mutation studies revealed the different TSS on different promoters. Targeted mutation into the core promoter area revealed the important sequences which were necessary for transcription initiation.

In case of the identification of core promoter elements of a new organism, the follwoing steps can be undertaken :

1. DNA is isolated from the organism and, in case the genomic sequence of the specific organism is not readily available, the whole genome is sequenced.
2. Sequence homology is tested with other organisms using the sequence database, to identify protein-coding regions or exons.
3. The exons should be preceded by promoter regions. Therefore, plasmids are designed according to the region upstream of the exon sequences, and a reporter gene along with the antibiotic-resistance gene is inserted into the plasmids.
4. Transfection of the plasmids is carried out, followed by an incubation period when the plasmid gets integrated into the chromosome via homologous recombination.
5. The activity of the suppsoed 'promoter' region is studied by the expression level of the reporter gene and antibiotic resistance activity.
6. The plasmid can be designed to recombine at various locations upstream of the exon, in order to check which region is the most effective in carrying out transcription.
7. Also, site-specific mutations in the region upstream of the exon can reveal the TSS.

This is how the core promoter elements can be identified in a new organism.

(Footnote : The question says how to identify core promoter elements in a new organ. I presume this was a typing error, since promoter elements cannot be present in any other organ except the nucleus, where transcription takes place. Therefore in the answer 'organ' has been replaced with 'organism'.)