13.4 Eukaryotic Transcription Is Similar to Bacterial Transcription but Has Some Important Differences

Transcription in eukaryotes is similar to bacterial transcription in that it includes initiation, elongation, and termination, and the basic principles of transcription already outlined apply to eukaryotic transcription. However, there are some important differences. Eukaryotic cells possess three different RNA polymerases, each of which transcribes a different class of RNA and recognizes a different type of promoter. Thus, a generic promoter cannot be described for eukaryotic cells; rather, a promoter’s description depends on whether the promoter is recognized by RNA polymerase I, II, or III. Another difference is in the nature of promoter recognition and initiation. Many proteins take part in the binding of eukaryotic RNA polymerases to DNA templates, and the different types of promoters require different proteins.

Transcription and Nucleosome Structure

Transcription requires sequences on DNA to be accessible to RNA polymerase and other proteins. However, in eukaryotic cells, DNA is complexed with histone proteins in highly compressed chromatin (see Figure 11.4). How can the proteins necessary for transcription gain access to eukaryotic DNA when it is complexed with histones?

The answer to this question is that chromatin structure is modified before transcription so that the DNA is in a more open configuration and is more accessible to the transcription machinery. Several types of proteins have roles in chromatin modification. Acetyltransferases add acetyl groups to amino acids at the ends of the histone proteins, which destabilizes nucleosome structure and makes the DNA more accessible. Other types of histone modification also can affect chromatin packing. In addition, proteins called chromatin-remodeling proteins may bind to the chromatin and displace nucleosomes from promoters and other regions important for transcription. We will take a closer look at the role of changes in chromatin structure associated with gene expression in Chapter 17.

Promoters

A significant difference between bacterial and eukaryotic transcription is the existence of three different eukaryotic RNA polymerases, which recognize different types of promoters. In bacterial cells, the holoenzyme (RNA polymerase plus the sigma factor) recognizes and binds directly to sequences in the promoter. In eukaryotic cells, promoter recognition is carried out by accessory proteins that bind to the promoter and then recruit a specific RNA polymerase (I, II, or III) to the promoter.

One class of accessory proteins comprises general transcription factors, which, along with RNA polymerase, form the basal transcription apparatus—a group of proteins that assemble near the start site and are sufficient to initiate minimal levels of transcription. Another class of accessory proteins consists of transcriptional activator proteins, which bind to specific DNA sequences and bring about higher levels of transcription by stimulating the assembly of the basal transcription apparatus at the start site.

We will focus our attention on promoters recognized by RNA polymerase II, which transcribes the genes that encode proteins. A promoter for a gene transcribed by RNA polymerase II typically consists of two primary parts: the core promoter and the regulatory promoter.

Core Promoter

The core promoter is located immediately upstream of the gene (Figure 13.15) and is the site to which the basal transcription apparatus binds. The core promoter typically includes one or more consensus sequences. One of the most common of these sequences is the TATA box, which has the consensus sequence TATAAA and is located from −25 to −30 bp upstream of the start site. Additional consensus sequences that may be found in the core promoters of genes transcribed by RNA polymerase II are shown in Figure 13.15. These consensus sequences are recognized by transcription factors that bind to them and serve as a platform for the assembly of the basal transcription apparatus.

Figure 13.15: The promoters of genes transcribed by RNA polymerase II consist of a core promoter and a regulatory promoter that contain consensus sequences. Not all the consensus sequences shown are found in all promoters.

Regulatory Promoter

The regulatory promoter is located immediately upstream of the core promoter. A variety of different consensus sequences can be found in the regulatory promoters, and they can be mixed and matched in different combinations. Transcriptional activator proteins bind to these sequences and either directly or indirectly make contact with the basal transcription apparatus and affect the rate at which transcription is initiated. Transcriptional activator proteins also regulate transcription by binding to more-distant sequences called enhancers. The DNA between an enhancer and the promoter loops out, and so transcriptional activator proteins bound to the enhancer can interact with the basal transcription machinery at the core promoter. Enhancers will be discussed in more detail in Chapter 17.

Polymerase I and III Promoters

RNA polymerase I and RNA polymerase III each recognize promoters that are distinct from those recognized by RNA polymerase II. For example, promoters for small rRNA and tRNA genes, transcribed by RNA polymerase III, contain internal promoters that are downstream of the start site and are transcribed into the RNA.

Initiation

Transcription in eukaryotes is initiated through the assembly of the transcriptional machinery on the promoter. This machinery consists of RNA polymerase II and a series of transcription factors that form a giant complex consisting of 50 or more polypeptides. Assembly of the transcription machinery begins when regulatory proteins bind DNA near the promoter and modify the chromatin structure so that transcription can take place. These proteins and other regulatory proteins then recruit the basal transcriptional apparatus to the core promoter.

The basal transcription apparatus consists of RNA polymerase, a series of general transcription factors, and a complex of proteins known as the mediator (Figure 13.16). The general transcription factors include TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH, in which TFII stands for transcription factor for RNA polymerase II and the final letter designates the individual factor.

Figure 13.16: Transcription is initiated at RNA polymerase II promoters. Transcription is initiated when the TFIID transcription factor binds to the TATA box, followed by the binding of a preassembled holoenzyme containing general transcription factors, RNA polymerase II, and the mediator. TBP stands for TATA-binding protein.

RNA polymerase II and the general transcription factors assemble at the core promoter, forming a pre-initiation complex that is analogous to the closed complex seen in bacterial initiation. Recall that, in bacteria, the sigma factor recognizes and binds to the promoter sequence. In eukaryotes, the function of sigma is replaced by that of the general transcription factors. A first step in initiation is the binding of TFIID to the TATA box on the DNA template. TFIID consists of at least nine polypeptides. One of them is the TATA-binding protein (TBP), which recognizes and binds to the TATA consensus sequence. The TATA-binding protein binds to the minor groove and straddles the DNA as a molecular saddle (Figure 13.17), bending the DNA and partly unwinding it. Other transcription factors bind to additional consensus sequences in the core promoter and to RNA polymerase and position the polymerase over the transcription start site.

Figure 13.17: The TATA-binding protein (TBP) binds to the minor groove of DNA, straddling the double helix of DNA like a saddle.

After the RNA polymerase and transcription factors have assembled on the core promoter, conformational changes take place in both the DNA and the polymerase. These changes cause from 11 to 15 bp of DNA surrounding the transcription start site to separate, producing the single-stranded DNA that will serve as a template for transcription.

The single-stranded DNA template is positioned within the active site of RNA polymerase, creating a structure called the open complex. After the open complex has formed, the synthesis of RNA begins as phosphate groups are cleaved off nucleoside triphosphates and nucleotides are joined together to form an RNA molecule. As in bacterial transcription, RNA polymerase may generate and release several short RNA molecules in abortive transcription before the polymerase initiates the synthesis of a full-length RNA molecule. TRY PROBLEM 35

Elongation

After about 30 bp of RNA have been synthesized, the RNA polymerase leaves the promoter and enters the elongation stage of transcription. Many of the transcription factors are left behind at the promoter and can serve to quickly reinitiate transcription with another RNA polymerase enzyme.

The molecular structure of eukaryotic RNA polymerase II and how it functions during elongation have been revealed through the work of Roger Kornberg and his colleagues, for which Kornberg was awarded a Nobel Prize in chemistry in 2006. The RNA polymerase maintains a transcription bubble during elongation, in which about eight nucleotides of RNA remain base paired with the DNA template strand. The DNA double helix enters a cleft in the polymerase and is gripped by jawlike extensions of the enzyme (Figure 13.18). The two strands of the DNA are unwound and RNA nucleotides that are complementary to the template strand are added to the growing 3' end of the RNA molecule. As it funnels through the polymerase, the DNA-RNA hybrid hits a wall of amino acids and bends at almost a right angle; this bend positions the end of the DNA-RNA hybrid at the active site of the polymerase, and new nucleotides are added to the 3' end of the growing RNA molecule. The newly synthesized RNA is separated from the DNA and runs through another groove before exiting from the polymerase.

Figure 13.18: The structure of RNA polymerase II is a source of insight into its function. The DNA double helix enters the polymerase through a groove and unwinds. The DNA-RNA duplex is bent at a right angle, which positions the 3′ end of the RNA at the active site of the enzyme. New nucleotides are added to the 3′ end of the RNA.

Termination

The three eukaryotic RNA polymerases use different mechanisms for termination. RNA polymerase I requires a termination factor like the rho factor utilized in the termination of some bacterial genes. Unlike rho, which binds to the newly transcribed RNA molecule, the termination factor for RNA polymerase I binds to a DNA sequence downstream of the termination site.

RNA polymerase III ends transcription after transcribing a terminator sequence that produces a string of uracil nucleotides in the RNA molecule, like that produced by the rho-independent terminators of bacteria. Unlike rho-independent terminators in bacterial cells, however, RNA polymerase III does not require that a hairpin structure precede the string of Us.

The termination of transcription by RNA polymerase II is not at specific sequences. Instead, RNA polymerase II often continues to synthesize RNA hundreds or even thousands of nucleotides past the coding sequence necessary to produce the mRNA. As we will see in Chapter 14, the end of pre-mRNA is cleaved at a specific site, designated by a consensus sequence, while transcription is still taking place at the 3′ end of the molecule. Cleavage cuts the pre-mRNA into two pieces: the mRNA that will eventually encode the protein and another piece of RNA that has its 5′ end trailing out of the RNA polymerase (Figure 13.19). An enzyme (called Rat1 in yeast) attaches to the 5′ end of this RNA and moves toward the 3 end where RNA polymerase continues the transcription of RNA. Rat1 is a 5′→3′ exonuclease−an enzyme capable of degrading RNA in the 5′→3′ direction. Like a guided torpedo, Rat1 homes in on the polymerase, chewing up the RNA as it moves. When Rat1 reaches the transcriptional machinery, transcription terminates. Note that this mechanism is similar to that of rho-dependent termination in bacteria (see Figure 13.13), except that rho does not degrade the RNA molecule.

Figure 13.19: Termination of transcription by RNA polymerase II requires the Rat1 exonuclease. Cleavage of the pre-mRNA produces a 5′ end to which Rat1 attaches. Rat1 degrades the RNA molecule in the 5′→3′direction. When Rat1 reaches the polymerase, transcription is halted.