Transcription of Eukaryotic Genes

TRANSCRIPTION OF EUKARYOTIC GENES

The transcription of eukaryotic genes is a far more complicated process than transcription in prokaryotes. Eukaryotic transcription involves separate polymerases for the synthesis of rRNA, tRNA, and mRNA. In addition, a large number of proteins called transcription factors (TFs) are involved. TFs bind to distinct sites on the DNA either within the core promoter region, close (proximal) to it, or some distance away (distal). They are required both for the assembly of a transcription complex at the promoter and the determination of which genes are to be transcribed. [Note: Each eukaryotic RNA pol has its own promoters and TFs that bind core promoter sequences.] For TFs to recognize and bind to their specific DNA sequences, the chromatin structure in that region must be altered (relaxed) to allow access to the DNA. The role of transcription in the regulation of gene expression is discussed in Chapter 32.

A. Chromatin structure and gene expression

The association of DNA with histones to form nucleosomes affects the ability of the transcription machinery to access the DNA to be transcribed. Most actively transcribed genes are found in a relatively relaxed form of chromatin called euchromatin, whereas most inactive segments of DNA are found in highly condensed heterochromatin. [Note: The interconversion of these forms is called chromatin remodeling.] A major component of chromatin remodeling is the covalent modification of histones (for example, the acetylation of lysine residues at the amino terminus of histone proteins) as shown in Figure 30.11. Acetylation, mediated by histone acetyltransferases (HATs), eliminates the positive charge on the lysine, thereby decreasing the interaction of the histone with the negatively charged DNA. Removal of the acetyl group by histone deacetylases (HDACs) restores the positive charge and fosters stronger interactions between histones and DNA. [Note: The ATP-dependent repositioning of nucleosomes is also required to access DNA.]

Figure 30.11 Acetylation/deacetylation of a lysine residue in a histone protein. HAT = histone acetyltransferase; HDAC = histone deacetylase.

B. Nuclear RNA polymerases of eukaryotic cells

There are three distinct classes of RNA pol in the nucleus of eukaryotic cells. All are large enzymes with multiple subunits. Each class of RNA pol recognizes particular types of genes.

1. RNA polymerase I: This enzyme synthesizes the precursor of the 28S, 18S, and 5.8S rRNA in the nucleolus.

2. RNA polymerase II: This enzyme synthesizes the nuclear precursors of mRNA that are subsequently translated to produce proteins. RNA pol II also synthesizes certain small ncRNAs, such as snoRNA, snRNA and miRNA.

a. Promoters for RNA polymerase II: In some genes transcribed by RNA pol II, a sequence of nucleotides (TATAAA) that is nearly identical to that of the Pribnow box is found centered about 25 nucleotides upstream of the transcription start site. This core promoter consensus sequence is called the TATA, or Hogness, box. In the majority of genes, however, no TATA box is present. Instead, different core promoter elements such as Inr (initiator) or DPE (downstream promoter element) are present (Figure 30.12). [Note: No one consensus sequence is found in all core promoters.] Because these sequences are on the same molecule of DNA as the gene being transcribed, they are cis-acting. The sequences serve as binding sites for proteins known as general transcription factors (GTFs), which in turn interact with each other and with RNA pol II.

Figure 30.12 Eukaryotic gene cis-acting promoter and regulatory elements and their trans-acting general and specific transcription factors (GTF and STF, respectively). Inr = initiator; DPE = downstream promoter element.

b. General transcription factors: These are the minimal requirements for recognition of the promoter, recruitment of RNA pol II to the promoter, and initiation of transcription at a basal level (Figure 30.13A). GTFs are encoded by different genes, synthesized in the cytosol, and transit to their sites of action, and so are trans-acting. [Note: In contrast to the prokaryotic holoenzyme, eukaryotic RNA pol II does not itself recognize and bind the promoter. Instead, TFIID, a GTF containing TATA-binding protein and TATA-associated factors, recognizes and binds the TATA box (and other core promoter elements). TFIIF, another GTF, brings the polymerase to the promoter. The helicase activity of TFIIH melts the DNA, and its kinase activity phosphorylates polymerase, allowing it to clear the promoter.]

Figure 30.13 A. Association of the general transcription factors (TFIIs) and RNA polymerase II (RNA pol II) at the core promoters. [Note: The Roman numeral II denotes the TFs for RNA pol II.] B. Enhancer stimulation of transcription. CTF = CAAT box transcription factor; Sp1 = specificity factor-1.

c. Regulatory elements and transcriptional activators: Upstream of the core promoter are additional consensus sequences (see Figure 30.12). Those close to the core promoter (within 200 nucleotides) are the proximal regulatory elements, such as the CAAT and GC boxes. Those farther away are the distal regulatory elements such as enhancers. Proteins known as transcriptional activators or specific transcription factors (STFs) bind these regulatory elements. STFs bind to promoter proximal elements to regulate the frequency of transcription initiation, and to distal elements to mediate the response to signals such as hormones and regulate which genes are expressed at a given point in time. A typical protein-coding eukaryotic gene has binding sites for many such factors. [Note: STFs have two binding domains. One is a DNA-binding domain, the other is a transcription activation domain that recruits the GTFs to the core promoter as well as “coactivator” proteins such as the HAT enzymes involved in chromatin modification.]

Transcriptional activators bind DNA through a variety of motifs, such as the helix-loop-helix, zinc finger, and leucine zipper.

Figure 30.14 Some possible locations of enhancer sequences.

d. Role of enhancers in eukaryotic gene regulation: Enhancers are special DNA sequences that increase the rate of initiation of transcription by RNA pol II. Enhancers are typically on the same chromosome as the gene whose transcription they stimulate (Figure 30.13B). However, they can 1) be located upstream (to the 5 -side) or downstream (to the 3 -side) of the transcription start site, 2) be close to or thousands of base pairs away from the promoter (Figure 30.14), and 3) occur on either strand of the DNA. Enhancers contain DNA sequences called “response elements” that bind STFs (transcriptional activators). By bending or looping the DNA, these enhancer-binding proteins can interact with other transcription factors bound to a promoter and with RNA pol II, thereby stimulating transcription (see Figure 30.13B). [Note: Although silencers are similar to enhancers in that they also can act over long distances, they reduce gene expression.]

e. Inhibitors of RNA polymerase II: α-Amanitin, a potent toxin produced by the poisonous mushroom Amanita phalloides (sometimes called “the death cap”), forms a tight complex with RNA pol II, thereby inhibiting mRNA synthesis.

3. RNA polymerase III: This enzyme synthesizes tRNA, 5S rRNA, and some snRNA and snoRNA.

C. Mitochondrial RNA polymerase

Mitochondria contain a single RNA pol that more closely resembles bacterial RNA pol than the eukaryotic nuclear enzymes.