When transcription is terminated the core RNA polymerase is released. In order to start a new round of transcription, the core RNA polymerase has to be directed to bind at a promoter, defined as the specific DNA sequence where transcription is initiated. There are specific DNA binding factors that bind to promoters and to RNA polymerase. That's how they direct RNA polymerase to the place where transcription has to start. These factors bind first to core polymerase forming the second form of RNA polymerase called the holoenzyme.
The binding parameters of the E. coli core polymerase and the holoenzyme have been studied in detail. In E. coli cells there are several different versions of holoenzyme. Each one contains a different initiation factor that binds to a different series of promoters. The most common initiation factor is called σ70 (sigma-70) and it binds to most of the promoters in the cell.
The steps in transcription initiation are shown in the figure. First, holoenzyme consisting of core polymerase + σ70, binds non-specifically to any stretch of DNA. It then moves along the DNA in a one-dimensional search until it finds a promoter sequence. This is followed by a local unwinding of the DNA and synthesis of a short piece of DNA.
Subsequent steps (not shown) require the dissociation of the initiation factor (σ70) and the formation of an elongation complex. RNA polymerase is then free to leave the promoter region and move down the gene making RNA.
The same kinds of parameters that we discussed yesterday are used to describe RNA polymerase binding [see DNA Binding Proteins and Repression of the lac Operon]. The core RNA polymerase by itself binds DNA non-specifically with an association, or binding, constant (Ka) of 1010 M-1. This is very tight binding for a DNA binding protein. Once bound to DNA the core RNA polymerase dissociates very slowly (t1/2 = 60 minutes).
The holoenzyme can also bind non-specifically. In this case the association constant is 5 × 106 M-1 and the complex dissociates rapidly (t1/2 = 3 seconds). The holoenzyme binds specifically to promoter sequences with an association constant of 2 × 1011 M-1 and t1/2 = 2 to 3 hours. Thus, the interaction of the initiation factor with core RNA polymerase has two effects: it decreases the affinity for random stretches of DNA and increases the affinity for the promoter sequence.
A typical E. coli cell contains about 5000 molecules of RNA polymerase. When the cells are growing rapidly, 2500 molecules will be bound to genes in transcription complexes. Another 1250 will be in initiation complexes of various sorts and most of the remaining RNA polymerase molecules (1200) will be bound to DNA non-specifically. Only a small number (~50) will be free in the cytoplasm.
Since the holoenzyme molecules are capable of initiating transcription on their own, a small number of the non-specifically bound molecules will accidentally transcribe short stretches of DNA. These spurious transcripts don't usually cause a problem since they are quite rare. Nevertheless, their presence means that much of the intergenic DNA in the E. coli genome is transcribed at one time or another.
Eukaryotic cells contain three different kinds of RNA polymerases [Eukaryotic RNA Polymerases]. Each one is much more complex that the bacterial enzymes but the principles of transcription initiation are the same.
In eukaryotes there are about a dozen general initiation factors for each of the different RNA polymerases. The ones for RNA polymerase II—the enzyme that transcribes protein-encoding genes—are called transcription factor IID (TFIID) etc. All of the factors are required for specific RNA polymerase binding at a promoter site and all of them associate with the core RNA polymerase to form a large holoenzyme complex. The eukaryotic general initiation factors do the same thing for eukaryotic core polymerase as the bacterial ones do for the bacterial RNA polymerase; they convert the complex to a specific DNA binding protein and lower its affinity for binding non-specifically.
As is the case in bacteria, a substantial number of holoenzyme complexes will be bound non-specifically to DNA at any one time. The proportion is much, much higher in mammalian cells because of the presence of so much junk DNA in the genome. This has the effect of soaking up a lot of holoenzyme complexes.
Since the holenzyme complexes, like those in bacteria, are capable of initiating basal levels of transcription, we should not be surprised to find spurious transciption in all parts of the genome. These transcript will be rare but they will come from any site where RNA polymerase holoenzme can bind.
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