Primary information about promoters
To turn a gene into a protein product, at least two general steps are required:
- the gene is transcribed, spliced and processed to form mRNA, and
- the mRNA is translated into a polypeptide.
Transcription is a controlled process. While multiple DNA regions are involved, the promoter is the main determinant for the initiation of transcription and modulation of levels and timing of gene expression.
Promoters in eukaryotic organisms- e.g. plants, animals- comprise multiple elements, some of which are found in nearly all promoters. These include:
- CAAT box. A consensus sequence close to -80 bp from the start point (+1). It plays an important role in promoter efficiency, by increasing its strength, and it seems to function in either orientation. This box is replaced in plants by a consensus sequence called the AGGA box;
- TATA box. A sequence usually located around 25 bp upstream of the start point. The TATA box tends to be surrounded by GC rich sequences. The TATA box binds RNA polymerase II and a series of transcription factors (TFIIX, X being a letter that identifies an individual transcription factor) to form an initiation complex;
- GC box. A sequence rich in guanidine (G) and cytidine (C) nucleotides, is usually found in multiple copies in the promoter region, normally surrounding the TATA box; and
- CAP site. A transcription initiation sequence or start point defined as +1, at which the transcription process actually starts.
|Conserved eukaryotic promoter elements||Consensus sequence|
RNA polymerase II is the enzyme that transcribes a gene into RNA. It works in conjunction with other transcription factors that recognize signals embodied in the promoter region. RNA polymerase II starts its “journey” at the TATA region where it binds and travels along the DNA until it reaches the CAP site where the actual synthesis of RNA starts. The transcription process only takes place in the downstream direction, from 5′ (left) to 3′ (right).
These elements are normally regarded as constituents of the promoter region itself, but depending on the scope of definition of a promoter in a patent or patent application, and whether the definition is expressed in functional or structural terms, other elements may be included as part of a promoter region.
Enhancers, for example, are elements located at variable distances from the promoter “itself” and contain several closely arranged sequence elements that bind to transcription factors. These elements enhance the activity of a promoter and are orientation-independent with respect to the promoter and can be upstream or downstream of a promoter (e.g., such as within intron sequences of a gene). There is currently a high interest in studying and isolating enhancers, which can be successfully attached to heterologous promoter regions to increase transcriptional activity and in some cases to provide additional levels of control (e.g., to confer tissue-specific or stage-specific expression of a gene).
Promoters of prokaryotic organisms, e.g., such as bacteria, have similar elements as the eukaryotic promoters although there are a few basic differences. Prokaryotic promoters contain at least three conserved features defining the region where the RNA polymerase binds:
- the start point, defined as +1;
- the TATA box is located at -10 position to the start point; in contrast to the -35 bp in eukaryotic promoters; and
- the TTGACA sequence, also called the -35 sequence, located around 35 bp upstream of the start point.
An additional feature, much more common in prokaryotic organisms, is that a promoter serves to initiate the transcription of multiple structural genes that are immediately adjacent to it. This arrangement is called an operon. A single transcribed mRNA is translated into several proteins whose functions are interrelated. In operons, promoters have adjacent, juxtaposed or interspersed regulatory sites to which regulatory proteins bind. In eukaryotic promoters, the regulatory sites are spread out over a longer distance.
There are two modes of regulation of the initiation of transcription in operons:
- Positive control mode, where the interaction between the regulatory protein and regulatory region on the DNA turn the transcription on. The genes are off by default and are turned on by the activators. Transcription factors interact with the RNA polymerase and assist the enzyme in initiating transcription at the promoter. This positive fashion of controlling gene expression is more common in eukaryotes than in prokaryotes.
- Negative control mode, where the interaction turns the genes off. In this case, a repressor protein binds the operator, a DNA sequence of approximately 20 to 25 nucleotides, which is next to the promoter or juxtaposed, and prevents the RNA polymerase from initiating transcription. To switch on the system, small molecules called inducers trigger the production of proteins by binding to the repressor protein and changing its conformation. This change alters the operator-repressor interaction, so that the repressor can no longer remain attached to the operator. Negative control is widely used among prokaryotes, which need to respond swiftly to changes in the environment.
One of the best-studied operon systems is the lac operon from Escherichia coli (E. coli). Since its discovery in the 1960s, other operon systems have been extensively studied in other organisms and are currently being adapted to plant systems with the aim of tightly regulating the expression of genes in transgenic plants.