Transcription In Eukaryotes

The research revolving around transcription initiation, especially in eukaryotes, is a hot spot for all areas of biology and even in chemistry. The first part of initiation is aligning the RNA polymerase to the +1 site of the gene. This is not done by RNA polymerase alone, but by a multitude of proteins that associate with both the DNA and the polymerase. These are done by the TATA-Binding protein (TPB), TPB-associated factors (TAFs), transcription factors (TFIIs), and an activated mediator (6-7).

The mediator is activated through different proteins depending on the gene being transcribed. When the mediator is activated by these proteins, the mediator stimulates the RNA polymerase pre-initiation complex. To create the pre-initiation complex, the TBP associates to the TATA site on the promoter and recruits TAFs. The activated mediator associates with TFIIE, RNA polymerase II, and the TAFs. Also connected to the TBP is TFIIB which associates with the promoter and RNA polymerase. TFIIF also coordinates the association of RNA polymerase and the promoter. Finally part of this complex is TFIIH which associates with TFIIE and the promoter (6). This forms the complete pre-initiation complex. TFIIE and TFIIH finalize the complex to stabilize it which results in the melting of the DNA. These two factors are also involved with the transition from initiation into elongation, where they remove the transcription factors that have already completed their roles in initiation (8).

The surface of RNA polymerase II is highly negative. The cleft, the wall, the active center, and the “saddle” are positively charged (9). Since DNA is negatively charged, this positive change probably allows the negatively charged DNA to be directed into these regions of this protein.

Due to recent RNA polymerase II models a greater insight into the specific activity of the ten different subunits can be determined. The ten subunits are Rpb1 through Rpb12 (there is no Rpb4 or Rpb7). These subunits work together to make a very efficient protein.

RNA polymerase II can be separated into four different modules that can move relative to each other. The largest module, called the “core” module, contains Rpb1 and Rpb2 that together form the active center of the polymerase. Contained in the Rpb1 subunit is a highly conserved region called the bridge helix. Rpb1 contains the active site and Rpb2 contains the “hybrid-binding” region. Also in this first module are the subunits Rpb3, Rpb10, Rpb11, and Rpb12. The second module is also called the “jaw-lobe” module. This module contains the “upper-jaw” which is made of Rpb1 and Rpb9. Also contained in this module is the “lobe” which is made of part of Rpb2. The “shelf” module is the third mobile part of this polymerase, which is made of Rpb5 and makes up the lower part of the jaw. Part of this module is the “assembly” domain (by Rpb5 and Rpb6). The “foot” and “cleft” regions from Rpb1 also make up this module. The final mobile module is the “clamp” which is formed by the NH2- and COOH-terminus of Rpb1 and the COOH-terminal part of Rpb2 (9).

The clamp seems to open and close which could permit the entry of promoter DNA. It is also suggested that this module works to identify the DNA-RNA hybrid and to separate the DNA and RNA strands at the upstream end of the transcription bubble. At the base of the clamp is the cleft region of Rpb1, the anchor of Rpb2 and Rpb6. At this region there are a set of “switch” regions that are flexible and undergo conformational changes during the transition to a transcribing state. A few of these switches closes the clamp in the presence of a DNA-RNA hybrid (9).

Three loops extend from the clamp into the active center region. The loop nearest to the active center acts a “rudder” to help facilitate the separation of RNA from DNA as well as maintaining the upstream end of the RNA-DNA hybrid. The other two loops correspond to the “lid” and “zipper” may also participate in these functions as well. Also part of this active center are two metal ions that mark the very center of the active site and are coordinated largely by aspartates.

Every named part of the polymerase has a specific function or functions for RNA polymerase. The jaw and the lobe are both take part in the guiding of DNA through the protein. “Following this path, the DNA contacts the jaw domain of Rpb9, fits into a concave surface of the Rpb2 lobe, and passes over the saddle, where it is surrounded by switch 2, switch 3, the rudder and the flap loop (9).” As the template single strand emerges during late initiation, it can bind to nearby sites in the active center, which include both the cleft floor and the wall (9). The bridge plays an important role of exposing the next open site on the template to be base paired and polymerized as well as separating the RNA-DNA hybrid (10). Any subunits not mentioned as being part of any specific function contribute to the structural integrity of RNA polymerase.

The studies by the Kornberg group have dramatically increased the knowledge of transcription in eukaryotes. Now there is a much clearer picture into how mRNA transcripts are made. This has opened a window to many different areas of biology, including stem cell research.