In prokaryotes, transcription of genes is regulated by the binding of repressor proteins to specific sites in the promoter of the gene. When there is no repressor bound to the promoter, RNA polymerase will bind and initiate transcription of the gene into RNA. If the repressor is bound to the operator site within the promoter, RNA polymerase cannot bind and no RNA will be produced.
The examples I gave for genes involved in lactose utilization and tryptophan synthesis illustrate the principle of how proteins that bind to specific sites or sequences on the DNA molecule can regulate gene expression. The proteins that regulate gene expression recognize a very specific part of the gene and will bind to the DNA only at that defined sequence of bases in the DNA molecule. It is this sequence of bases that imparts the specificity to this regulation. In other words, the repressor that regulates beta-galactosidase expression only binds to the promoter of the beta-galactosidase gene. It will not bind to the promoters of other genes.
How can this system of regulation be manipulated to alter the way that genes are expressed?
First, consider what would happen if the DNA sequence that is recognized by the repressor was altered. The structure of the repressor protein allows it to bind to a specific DNA sequence. When the sequence of bases in the DNA is changed, the repressor will not bind and transcription of the gene will occur, regardless of whether lactose or tryptophan are present in the growth medium. Many mutants have been characterized with precisely this type of DNA sequence alteration in the repressor binding site.
See overhead of DNA sequence of lactose aperon.
The promoter region of this gene contains the elements that I have described before and also shows some features that I have not discussed. The operator region, to which the repressor binds, is at the site for the start of transcription. Upstream of this is the site for binding of RNA polymerase. The sequences at -10 and -35 are conserved in bacterial genes and allow binding of RNA polymerase to this site. The transcript starts at nucleotide +1. The initiation codon (ATG) for the first methionine of the protein is at +39 to +41. The first 38 bases of the RNA are not used to encode amino acids, but this non-translated leader sequence contains another important element. The Shine-Dalgarno sequence, named after the scientists who discovered it, is the site for binding of ribosomes to the mRNA to allow translation of the protein.
These elements are now well defined as specific sequences of bases in the DNA molecule. What happens if the promoter from the b-galactosidase gene, which is normally regulated by lactose, is now placed in front of another gene, Gene X? The RNA from Gene X will be produced when lactose is present in the medium, but will not be expressed in the absence of lactose. As long as there is no other form of regulation for this gene, then protein X will be made only when lactose is added to the growth medium for these bacteria.
See overhead.
This experiment demonstrates that DNA sequences containing specific elements for gene expression can be exchanged between genes and still maintain their function. The ability to recombine the functional elements of different genes (the modules I have described earlier) is of great importance in manipulating the expression of genes in biotechnology.
Now we will discuss the regulation of gene expression in eukaryotes. The first point to make is that the same principles that govern transcription in prokaryotes also apply to eukaryotes. Specific pieces of DNA of defined sequence perform specific functions in directing transcription. However, in contrast to the compact nature of bacterial promoters, eukaryote promoters, especially those in higher eukaryotes, are spread out over much larger pieces of DNA. There are also a number of differences in the processing of RNA that is used to make proteins.
As a consequence of this complex gene organization, some human genes that encode proteins of a normal size can cover more than 100,000 base pairs. A gene that is required for normal copper metabolism in humans encodes a protein of approximately 1,500 amino acids. This requires a minimum of 4,500 base pairs (3 bp per amino acid x 1,500 amino acids) and the mRNA is about 8,000 bases in size. However, the gene that encodes this protein contains many introns and is about 135,000 base pairs in size. Mutations in this gene result in a condition known as Menkes disease. (See overhead)
Promoters of eukaryote genes perform the same functions as in bacterial genes.
See overhead.
The RNA polymerase that is responsible for transcription of eukaryote mRNAs, together with other general transcription factors, binds to the promoter of this type of gene, just upstream of the site where trancription starts. Other regulatory proteins bind to specific DNA sequences in the promoter region, usually upstream of the site where transcription starts. These other regulatory proteins interact with the general transcription machinery assembled at the TATA box to stimulate or modulate transcription of that gene.
As with bacterial genes, the specific DNA sequences in the promoter of a gene are the elements that direct the expression of that gene. If these DNA sequences are altered or deleted, then the pattern of gene expression will be altered. As with promoters of bacterial genes, promoters of eukaryote genes can also be exchanged and still mantain their function.
Unlike bacterial genes, these elements that regulate transcription are not tightly compressed into a small stretch of DNA, but can be spread out over tens of thousands of base pairs, and can be before, after, or in the middle of the transcription unit.
In summary, there are a number of important points to remember about the role that promoters play in the critical regulation of gene expression.
However, there are a number of important differences between prokaryote and eukaryote genes that must be remembered.
While there are many conceptual similarities between prokaryotes and eukaryotes in gene structure and gene regulation, there are many important functional differences in the details of how these operate in different organisms. One consequence of these differences in the details of how genes function is that if a gene is transferred from one species to another, it may not be expressed. If the gene is moved between two closely related species, it may function correctly (i.e. be expressed in the correct time and place). However, as genes are moved between species that are not closely related, it becomes more likely that the transferred gene will not be expressed.