Figure 6–16 Consensus sequences found in the vicinity of eukaryotic RNA polymerase II start points. The name given to each consensus sequence (first column) and the general transcription factor that recognizes it (last column) are indicated. N indicates any nucleotide, and two nucleotides separated by a slash indicate an equal probability of either nucleotide at the indicated position. In reality, each consensus sequence is a shorthand representation of a histogram similar to that of Figure 6–12.
For most RNA polymerase II transcription start points, only two or three of the four sequences are present. For example, many polymerase II promoters have a TATA box sequence, but those that do not typically have a “strong” INR sequence. Although most of the DNA sequences that influence transcription initiation are located upstream of the transcription start point, a few, such as the DPE shown in the figure, are located in the transcribed region.
seven-amino-acid sequence, which extend from the RNA polymerase core structure. During transcription initiation, the serine located at the fifth position in the repeat sequence (Ser5) is phosphorylated by TFIIH, which contains a protein kinase in one of its subunits (see Figure 6–15D and E). The polymerase can then disengage from the cluster of general transcription factors. During this process, it undergoes a series of conformational changes that tighten its interaction with DNA, and it acquires new proteins that allow it to transcribe for long distances, in some cases for many hours, without dissociating from DNA.
Once the polymerase II has begun elongating the RNA transcript, most of the general transcription factors are released from the DNA so that they are available to initiate another round of transcription with a new RNA polymerase molecule. As we see shortly, the phosphorylation of the tail of RNA polymerase II has an additional function: it causes components of the RNA-processing machinery to load onto the polymerase and thus be positioned to modify the newly transcribed RNA as it emerges from the polymerase.
Polymerase II Also Requires Activator, Mediator, and Chromatin-Modifying Proteins
Studies of RNA polymerase II and its general transcription factors acting on DNA templates in purified in vitro systems established the model for transcription initiation just described. However, as discussed in Chapter 4, DNA in eukaryotic cells is packaged into nucleosomes, which are further arranged in higher-order chromatin structures. As a result, transcription initiation in a eukaryotic cell is more complex and requires more proteins than it does on purified DNA. First, gene regulatory proteins known as transcriptional activators must bind to specific sequences in DNA (called enhancers) and help to attract RNA polymerase II to the start point of transcription (Figure 6–18). We discuss the role of these activators in Chapter 7, because they are one of the main ways in which cells regulate expression of their genes. Here we simply note that their presence on DNA is required for transcription initiation in a eukaryotic cell. Second, eukaryotic transcription initiation in vivo requires the presence of a large protein complex known as Mediator, which allows the activator proteins to communicate properly with the polymerase II and with the general transcription factors. Finally, transcription initiation in a eukaryotic cell typically requires the recruitment of chromatin-modifying enzymes, including chromatin remodeling complexes and
Figure 6–17 Three-dimensional structure of TbP (TATA-binding protein) bound to DNA. The TBP is the subunit of the general transcription factor TFIID that is responsible for recognizing and binding to the TATA box sequence in the DNA (red). The unique DNA bending caused by TBP—kinks in the double helix separated by partly unwound DNA—is thought to serve as a landmark that helps to attract the other general transcription factors (Movie 6.4). TBP is a single polypeptide chain that is folded into two very similar domains (blue and green). (Adapted from J.L. Kim et al., Nature 365:520–527, 1993. With permission from Macmillan Publishers Ltd.) start of (binding site for