Figure 6–15 Initiation of transcription of a eukaryotic gene by RNA polymerase II. To begin transcription, RNA polymerase requires several general transcription factors. (A) The promoter contains a DNA sequence called the TATA box, which is located 25 nucleotides away from the site at which transcription is initiated. (B) Through its subunit TBP, TFIID recognizes and binds the TATA box, which then enables the adjacent binding of TFIIB (C). For simplicity the DNA distortion produced by the binding of TFIID (see Figure 6–17) is not shown. (D) The rest of the general transcription factors, as well as the RNA polymerase itself, assemble at the promoter. (E) TFIIH then uses energy from ATP hydrolysis to pry apart the DNA double helix at the transcription start point, locally exposing the template strand. TFIIH also phosphorylates RNA polymerase II, changing its conformation so that the polymerase is released from the general factors and can begin the elongation phase of transcription. As shown, the site of phosphorylation is a long C-terminal polypeptide tail, also called the C-terminal domain (CTD), that extends from the polymerase molecule. The assembly scheme shown in the figure was deduced from experiments performed in vitro, and the exact order in which the general transcription factors assemble on promoters probably varies from gene to gene in vivo. The general transcription factors are highly conserved; some of those from human cells can be replaced in biochemical experiments by the corresponding factors from simple yeasts.
Figure 6–14 Structural similarity between a bacterial RNA polymerase and a eukaryotic RNA polymerase II. Regions of the two RNA polymerases that have similar structures are indicated in green. The eukaryotic polymerase is larger than the bacterial enzyme (12 subunits instead of 5), and some of the additional regions are shown in gray. The blue spheres represent Zn atoms that serve as structural components of the polymerases, and the red sphere represents the Mg atom present at the active site, where polymerization takes place. The RNA polymerases in all modern-day cells (bacteria, archaea, and eukaryotes) are closely related, indicating that the basic features of the enzyme were in place before the divergence of the three major branches of life. (Courtesy of
P. Cramer and R. Kornberg.) start of transcription TATA box (D) UTP, ATP CTP, GTP FACTOR RELEASE causes a large distortion in the DNA of the TATA box (Figure 6–17). This distortion is thought to serve as a physical landmark for the location of an active promoter in the midst of a very large genome, and it brings DNA sequences on both sides of the distortion closer together to allow for subsequent protein assembly steps. Other factors then assemble, along with RNA polymerase II, to form a complete transcription initiation complex (see Figure 6–15). The most complicated of the general transcription factors is TFIIH. Consisting of nine subunits, it is nearly as large as RNA polymerase II itself and, as we shall see shortly, performs several enzymatic steps needed for the initiation of transcription.
After forming a transcription initiation complex on the promoter DNA, RNA polymerase II must gain access to the template strand at the transcription start point. TFIIH, which contains a DNA helicase as one of its subunits, makes this step possible by hydrolyzing ATP and unwinding the DNA, thereby exposing the template strand. Next, RNA polymerase II, like the bacterial polymerase, remains at the promoter synthesizing short lengths of RNA until it undergoes a series of conformational changes that allow it to move away from the promoter and enter the elongation phase of transcription. A key step in this transition is the addition of phosphate groups to the “tail” of the RNA polymerase (known as the CTD or C-terminal domain). In humans, the CTD consists of 52 tandem repeats of a subunit of TFIID C/T C/T A N T/A C/T C/T TFIID