MoBio Transcription Mechanisms in Eukaryotes Chapter 4

In eukaryotes, there are three classes of RNA polymerases: I, II and III. This section will focus on the RNA polymerase II (Pol II), which is involved in the transcription of all protein genes. Transcription by RNA Pol I and Pol III is discussed in Section I.

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Figure 4-E-1. Structure of the human TBP core domain complexed with DNA as determined by x-ray crystallography. The DNA includes the TATA element. PDB ID = 1CDW.

Initiation

RNA Pol II does not contain a subunit similar to the prokaryotic σ factor, which can recognize the promoter and unwind the DNA double helix. In eukaryotes, these two functions are carried out by a set of proteins called general transcription factors. The RNA Pol II is associated with six general transcription factors, designated as TFIIA, TFIIB, TFIID, TFIIE, TFIIF and TFIIH, where "TF" stands for "transcription factor" and "II" for the RNA Pol II.

TFIID consists of TBP (TATA-box binding protein) and TAFs (TBP associated factors). The role of TBP is to bind the core promoter (Figure 4-E-1). TAFs may assist TBP in this process. In human cells, TAFs are formed by 12 subunits. One of them, TAF250 (with molecular weight 250 kD), has the histone acetyltransferase activity, which can relieve the binding between DNA and histones in the nucleosome.

The transcription factor which catalyzes DNA melting is TFIIH. However, before TFIIH can unwind DNA, the RNA Pol II and at least five general transcription factors (TFIIA is not absolutely necessary) have to form a pre-initiation complex (PIC). The order of the PIC assembly is described in Figure 4-E-2.

Elongation

After PIC is assembled at the promoter, TFIIH can use its helicase activity to unwind DNA. This requires energy released from ATP hydrolysis. The DNA melting starts from about -10 bp. Then, RNA Pol II uses nucleoside triphosphates (NTPs) to synthesize a RNA transcript. During RNA elongation, TFIIF remains attached to the RNA polymerase, but all of the other transcription factors have dissociated from PIC.

The carboxyl-terminal domain (CTD) of the largest subunit of RNA Pol II is critical for elongation. In the initiation phase, CTD is unphosphorylated, but during elongation it has to be phosphorylated. This domain contains many proline, serine and threonine residues.

Termination

Eukaryotic protein genes contain a poly-A signal located downstream of the last exon. This signal is used to add a series of adenylate residues during RNA processing. Transcription often terminates at 0.5 - 2 kb downstream of the poly-A signal, but the mechanism is unclear.

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The role of regulatory transcription factors

In early 1990s, when the mystery of transcriptional regulation in prokaryotes have been largely unveiled, scientists still knew very little about the regulation mechanism in eukaryotes. The breakthrough came in 1996 when a number of research groups discovered that certain transcriptional coactivators are histone acetyltransferases (HATs). It has been known for some time that binding of transcriptional activators to the enhancer region, in most cases, is not sufficient to stimulate transcription. Certain co-activators are also required. Similarly, transcriptional repression often requires both repressor binding on the silencer element and the participation of co-repressor proteins. The precise role of these co-activators and co-repressors was not clear until 1996.

In eukaryotes, the association between DNA and histones prevents access of the polymerase and general transcription factors to the promoter. Histone acetylation catalyzed by HATs can relieve the binding between DNA and histones. Although a subunit of TFIID (TAF250 in human) has the HAT activity, participation of other HATs can make transcription more efficient. The following rules apply to most (but not all) cases:

Binding of activators to the enhancer element recruits HATs to relieve association between histones and DNA, thereby enhancing transcription.

Binding of repressors to the silencer element recruits histone deacetylases (denoted by HDs or HDACs) to tighten association between histones and DNA.

For more information, see Section G.