Mechanism of Activation of RNAP Complex:
Proteins identify and specify the location of the gene regulatory regions, and assemble one after another and one upon the other in predetermined fashion. In spite of the assembly of basal transcription apparatus, the RNAP fails to initiate transcription with efficiency to the demands of the cell. The assembly of the basal transcriptional apparatus often initiatesthe transcription, by self-activation or through interaction with the associated components.
Most important aspect of the initiation is that the enzyme should be conformationally induced to change from loose binding to tight binding to the DNA, then from tight binding closed complex to tight binding open complex form. This isomerization goes to and fro several times before it starts assembly of first few nucleotides till it produces 10 to 11 ntds long transcript on the templates, before it clears the promoter region.
Stimulation of RNAP complex to initiate transcription requires several general transcriptional factors such as Sp1 (GC binding), CTF or CPs (CAAT binding), OCTA 1(OCTAMER binding), NFs (nuclear factors). These factors bind to their sequence specific boxes, which are located in the upstream very nearby. The binding of the said factors leads to interaction with basal PIC and the enzyme is activated to undergo conformational changes to initiate transcription.
A similar situation is also found with upstream activator and enhancer binding factors, which are positioned –200 or more distance from the start. The said factors have several roles, such as facilitating the assembly TF complexes such as TF-II-B, which is the rate-limiting factor in the assembly as well as initiation. Most of these factors, which are bound to the upstream element such as activators or enhancers, interact with PIC or basal apparatus, directly or through certain co activators, this requires the looping of the DNA, a contortion, a strain on the transcriptional apparatus bound DNA.
Each of the activator or enhancer proteins, which exhibit initiation efficiency of 200 or more times the normal, contains a DNA binding domain and an activator domain.
DNA binding domain consists of two half sites and dimer proteins bind one to each sites.
Then activator region is brought into contact with basal transcriptional machinery.
The activator domains, in most of the cases, which have been studied extensively, contain protein with acidic amino acids, called acidic domain. Acidic domains or acid blob interact with great affinity with TAFs or TFII-B or some, specific component of the enzyme and activate the enzyme into conformation change into induced state.
Removal of positive charges from the interacting domain enhances the activation process. But removal of acidic domain fails to activate transcription.
Mutation in GAL-4 that changes acidic amino acids in the activator region fails to activate transcription.
When HIV infects, it produces a regulatory protein called ‘tat’. The ‘tat’ protein has two binding domains, one DNA binding domain and another RNA binding domain called TAR. As the transcription is initiated, 5’ end of the RNA is made, but transcription doesn’t proceed. When the ‘tat’ protein binds to DNA as well as to RNA, the binding of RNA activates the PIC-enzyme complex.
This has been established by using hybrid protein by cloning, where DNA binding domain of one protein (this can be of repressor protein), and activator domain from another9it can be from any protein), resulted in activating the transcription, which means all activating domains have same features, but DNA binding domains differ, for it they make a difference, which gene or genes to activated.
Some of the TFs s such as SP1 which are glutamine rich and CTFs which are Proline rich, they work as activators for they have acidic domains. Sp1 specifically interacts with TAF elements. Acidic blobs can also act or interact with TF-II (?) and it can interact with other components of TF II-D.