Transcription bubble
A transcription bubble is a molecular structure formed during the initialization of DNA transcription, when a limited portion of the DNA double helix is unwound, providing enough space for RNA polymerase to bind to the template strand and begin RNA synthesis. The transcription bubble size is usually 12 to 14 base pairs, which allows the incorporation of complementary RNA nucleotides by the enzyme with ease. The dynamics and structure of the transcription bubble are variable, and play a role in the regulation of gene expression at the transcriptional level. The formation of bubbles depends on the structure of chromatin, the DNA sequence, and transcription factor, including H3K27ac histone acetylation marks, SWI/SNF nucleosome remodeling, and TFIIH and sigma factors. While the evolutionary history cannot be completely confirmed, scientists have provided various models to explain the most likely progression of bubble evolution, tying it directly to the divergence of archaea, eukaryotes, prokaryotes, and bacteria from the last universal common ancestor. Many drugs, including chemotherapeutic and antibiotic compounds, target elements of the transcription bubble to regulate gene transcription.
Formation
The formation of a transcriptional bubble precedes RNA synthesis and is initialized by the binding of the RNA polymerase to a promoter site, followed by the unwinding of the DNA double helix. This exposes a portion of single-stranded DNA, allowing RNA to be synthesized using it as a template. As such, the formation of the transcription bubble depends heavily on promoter quality and RNAP search mechanisms.Prokaryotic initiation
In prokaryotes, three mechanisms of RNAP's promoter search have been observed to various extents: 1D sliding, intersegment transfer, and hopping. While the extent that each mechanism contributes is uncertain, mechanism which depends on 3D diffusion seem to outweigh 1D diffusion in vitro. However, due to the abundance of macromolecules found in living cells, 3D diffusion may be hindered, leading to a larger contribution of 1D diffusion than in vitro studies observe.Various sigma factors mediate the association and stability of RNAP binding at a promoter site. RNAP binding of the σ factor creates RNA polymerase holoenzyme, the "active" form of bacterial RNAP. Binding of RNAP forms the closed promoter complex which must then isomerize into the open promoter complex, driving the formation of the transcription bubble. Two broad classes of σ factors exist: σ54 and σ70. σ54 binds to consensus sequences at -12 and -24 from the transcription start site, and recruits RNAP to form a stable RPc which rarely isomerizes into an RPo. Meanwhile, σ70 class factors recruit RNAP at -10 and -35, forming the RPo spontaneously. The recruitment of σ70 is mediated by various activators which can promote the formation of the RPc. After the formation of the transcription bubble, the σ factors dissociate from holoenzyme complex, allowing RNAP to proceed along the DNA template strand to complete RNA synthesis alone. The progression of RNAP occurs simultaneously with the rewinding of single stranded DNA upstream from the enzyme and the unwinding of double stranded DNA downstream from the enzyme, resulting in the "movement" of the transcription bubble with the RNAP.
Eukaryotic initiation
In eukaryotes, the search for loci to open transcription bubbles occurs through the recruitment of general transcription factors to a promoter region and formation of the preinitiation complex. Once the PIC forms, the DNA duplex is melted, forming the transcription bubble. Of the enzymes involved, the TATA-binding protein binds to the TATA box and causes DNA bending that leads to melting of the promoter region. The ATP-dependent helicase activity of XPB, a subunit of TFIIH, is required for DNA duplex unwinding and the formation of the transcription bubble after the PIC forms.After about 25 base pairs of the DNA double strand are unwound, RNA synthesis takes place within the transcription bubble region. DNA regions in front of RNA polymerase II unwinds to accommodate the movement of the enzyme while DNA regions behind it simultaneously rewind to reform the double helix in a manner similar to that of prokaryotes.
RNAP carries out the majority of the steps during the transcription cycle, especially in maintaining the transcription bubble open for the complementary base pairing. Some steps of the transcription cycle that require more proteins, such as the Rpb4/7 complex and the elongation factor Transcription Factor IIS.
After initiation, RNAP moves downstream along the template strand. The net effect of each RNA extension step is that RNAP takes one nucleotide triphosphate, elongates the nascent RNA by one nucleotide, and generates a single pyrophosphate ion. This is an energetically favorable reaction with a free energy change of approximately −5.6 kcal/mol, allowing RNAP to go forward along its target template which by association moves the bubble forward as well.
Termination
Prokaryotic termination
In Escherichia coli, the process of transcription termination via dissociation of the RNA polymerase have been found to depend on 3 possible mechanisms: an interaction between the polymerase and an intrinsic terminator sequence found on the hairpin loops of completed RNA, the presence of the RNA-dependent termination factor Rho, and the ATP-dependent DNA translocase Mfd.Studies have found that the disruption of the RNAP-DNA transcription complex by termination factor Rho is inhibited for as long as the upstream DNA in the transcription bubble remain unpaired. Thus, the detachment of bacterial RNAP from DNA in a rho-dependent process is preceded by and depends on the re-annealing of DNA within the transcription bubble.
During rho-independent termination, the transcription of a hairpin loop on completed RNA, which serves as the intrinsic termination sequence, contributes to the collapse of the transcription bubble. This is followed by the detachment of RNAP from the template DNA and the re-annealing of DNA strands. This method of termination does not require the presence of the transcription bubble, as E.coli RNAP have been observed in vitro to release the completed RNA transcript while using single-stranded DNA templates.
The third process of termination, involving DNA translocase Mfd, affects primarily transcription bubbles which have stalled in the presence of DNA damage. The presence of Mfd in the transcription bubble forces the downstream movement of RNAP without the addition of nucleoside triphosphates, inducing the re-annealing of the DNA in the transcription bubble and the detachment of both the RNAP and the nascent RNA.
Transcription bubble termination in E. coli is regulated by a variety of transcription factors. One such factor is NusG, a ribosomal protein that enhances the efficiency of Rho-dependent termination by aiding Rho recognition of termination sequences. NusG action is mandatory in situations where RNA release has to be performed in a small window of time.
Eukaryotic termination
Transcription termination by eukaryotic RNA polymerase I requires transcription termination factors similar to rho-dependent termination in prokaryotes. In mice, repeated terminators encoded on DNA are exposed as single-stranded binding sites for protein TTF-I once they are reached by the transcription bubble. The complex produced by the terminator and TTF-I binding then induces the release of the transcript. RNA Polymerase II is terminated through direct binding of the 3′-end cleavage and polyadenylation complex to the enzyme, which then releases the transcribed RNA. The recruitment of the CPA complex to the transcription bubble is induced by the transcription of a Poly-A signal on the nascent RNA.In both cases, RNA cleavage and release occurs before the dissociation of the polymerase from the transcription bubble. Thus, the integrity of the transcription bubble is temporarily preserved after the initiation of termination. Two models have been proposed to explain the process of polymerase dissociation after RNA release for both polymerases. The first is the torpedo model, in which the polymerase continues to synthesize RNA after the release of the nascent RNA. Exonuclease activity then degrades the new RNA strand, destabilizing RNA polymerase and achieving its dissociation from the transcription bubble. The second mechanism, the allosteric model, proposes that transcription of the poly A sequence near the end of nascent RNAs causes gradual dissociation of other transcription factors from the transcription bubble, causing a chain effect that eventually collapses the transcription bubble thorough destabilization.