Julianne Zedalis; John Eggebrecht

Learning Objectives

In this section, you will explore the following questions:

What are the steps in eukaryotic transcription?
What are the structural and functional similarities and differences among the three RNA polymerases?

Connection for AP^® Courses

As expected, transcription in eukaryotes is more complex than transcription in prokaryotes. First, transcription in eukaryotes involves one of three types of RNA polymerase, depending on the gene being transcribed. Second, the initiation of transcription involves the binding of several transcription factors to complex promoters which are usually located upstream of the gene being copied. Transcription factors can either activate or inhibit gene expression. Termination of transcription involves the RNA polymerases.

Information presented and the examples highlighted in the section support concepts outlined in Big Idea 3 of the AP^® Biology Curriculum Framework. The Learning Objectives listed in the Curriculum Framework provide a transparent foundation for the AP^® Biology course, an inquiry-based laboratory experience, instructional activities, and AP^® Exam questions. A Learning Objective merges required content with one or more of the seven Science Practices.

Big Idea 3	Living systems store, retrieve, transmit and respond to information essential to life processes.
Enduring Understanding 3.A	Heritable information provides for continuity of life.
Essential Knowledge	3.A.1 DNA, and in some cases RNA, is the primary source of heritable information.
Science Practice	6.5 The student can evaluate alternative scientific explanations.
Learning Objective	3.1 The student is able to construct scientific explanations that use the structures and mechanisms of DNA and RNA to support the claim that DNA and, in some cases, that RNA are the primary sources of heritable information.

Teacher Support

Ask students if they expect transcription to be different in eukaryotes. Ask students to explain their answers. Review the major difference between prokaryotes and eukaryotes, the presence of the nucleus.

Explain that the process follows the same general order, but there are major differences. The initiation of transcription in eukaryotes is more complicated. Polycistronic mRNAs exist in eukaryotes although they are very rare. Remind students, if applicable, that they have encountered nuclear transcription factors when you studied cell signaling. Point out the useful role of α-amanitin in differentiating between RNA polymerases. Remind students that the final product of a gene can be an RNA molecule, for example an rRNA molecule, or a polypeptide.

Ask students to prepare a timeline of transcription in eukaryotes and compare it to their timeline of transcription in prokaryotes. Teach students a simple way to remember which are the introns (in the trash) and which are the exons (expressed).

RNA polymerases in prokaryotes and eukaryotes have the same general functions; however, they are different enzymes. The numbering of RNA polymerases in eukaryotes does not reflect the order of activity. Pol II is the best studied polymerase, most likely because it transcribes mRNA.

Ribosome assembly takes place in the nucleolus which means that the ribosomal proteins are synthesized in the cytoplasm and re-enter the nucleus.

The Science Practice Challenge Questions contain additional test questions for this section that will help you prepare for the AP exam. These questions address the following standards:
[APLO 3.3][APLO 3.22][APLO 2.36][APLO 1.14][APLO 2.22][APLO 4.5]

Prokaryotes and eukaryotes perform fundamentally the same process of transcription, with a few key differences. The most important difference between prokaryotes and eukaryotes is the latter’s membrane-bound nucleus and organelles. With the genes bound in a nucleus, the eukaryotic cell must be able to transport its mRNA to the cytoplasm and must protect its mRNA from degrading before it is translated. Eukaryotes also employ three different polymerases that each transcribe a different subset of genes. Eukaryotic mRNAs are usually monogenic, meaning that they specify a single protein.

Initiation of Transcription in Eukaryotes

Unlike the prokaryotic polymerase that can bind to a DNA template on its own, eukaryotes require several other proteins, called transcription factors, to first bind to the promoter region and then help recruit the appropriate polymerase.

The Three Eukaryotic RNA Polymerases

The features of eukaryotic mRNA synthesis are markedly more complex those of prokaryotes. Instead of a single polymerase comprising five subunits, the eukaryotes have three polymerases that are each made up of 10 subunits or more. Each eukaryotic polymerase also requires a distinct set of transcription factors to bring it to the DNA template.

RNA polymerase I is located in the nucleolus, a specialized nuclear substructure in which ribosomal RNA (rRNA) is transcribed, processed, and assembled into ribosomes (Table 15.1). The rRNA molecules are considered structural RNAs because they have a cellular role but are not translated into protein. The rRNAs are components of the ribosome and are essential to the process of translation. RNA polymerase I synthesizes all of the rRNAs except for the 5S rRNA molecule. The “S” designation applies to “Svedberg” units, a nonadditive value that characterizes the speed at which a particle sediments during centrifugation.

Locations, Products, and Sensitivities of the Three Eukaryotic RNA Polymerases

RNA Polymerase	Cellular Compartment	Product of Transcription	α-Amanitin Sensitivity
I	Nucleolus	All rRNAs except 5S rRNA	Insensitive
II	Nucleus	All protein-coding nuclear pre-mRNAs	Extremely sensitive
III	Nucleus	5S rRNA, tRNAs, and small nuclear RNAs	Moderately sensitive

Table 15.1

RNA polymerase II is located in the nucleus and synthesizes all protein-coding nuclear pre-mRNAs. Eukaryotic pre-mRNAs undergo extensive processing after transcription but before translation. For clarity, this module’s discussion of transcription and translation in eukaryotes will use the term “mRNAs” to describe only the mature, processed molecules that are ready to be translated. RNA polymerase II is responsible for transcribing the overwhelming majority of eukaryotic genes.

RNA polymerase III is also located in the nucleus. This polymerase transcribes a variety of structural RNAs that includes the 5S pre-rRNA, transfer pre-RNAs (pre-tRNAs), and small nuclear pre-RNAs. The tRNAs have a critical role in translation; they serve as the adaptor molecules between the mRNA template and the growing polypeptide chain. Small nuclear RNAs have a variety of functions, including “splicing” pre-mRNAs and regulating transcription factors.

A scientist characterizing a new gene can determine which polymerase transcribes it by testing whether the gene is expressed in the presence of a particular mushroom poison, α-amanitin (Table 15.1). Interestingly, α-amanitin produced by Amanita phalloides, the Death Cap mushroom, affects the three polymerases very differently. RNA polymerase I is completely insensitive to α-amanitin, meaning that the polymerase can transcribe DNA in vitro in the presence of this poison. In contrast, RNA polymerase II is extremely sensitive to α-amanitin, and RNA polymerase III is moderately sensitive. Knowing the transcribing polymerase can clue a researcher into the general function of the gene being studied. Because RNA polymerase II transcribes the vast majority of genes, we will focus on this polymerase in our subsequent discussions about eukaryotic transcription factors and promoters.

Structure of an RNA Polymerase II Promoter

Eukaryotic promoters are much larger and more complex than prokaryotic promoters, but both have a TATA box. For example, in the mouse thymidine kinase gene, the TATA box is located at approximately -30 relative to the initiation (+1) site (Figure 15.10). For this gene, the exact TATA box sequence is TATAAAA, as read in the 5' to 3' direction on the nontemplate strand. This sequence is not identical to the E. coli TATA box, but it conserves the A–T rich element. The thermostability of A–T bonds is low and this helps the DNA template to locally unwind in preparation for transcription.

Illustration shows a series of transcription factors binding to the promoter, which is upstream of the gene. After all of the transcription factors are bound, RNA polymerase binds as well.

Figure 15.10 A generalized promoter of a gene transcribed by RNA polymerase II is shown. Transcription factors recognize the promoter. RNA polymerase II then binds and forms the transcription initiation complex.

Visual Connection

An illustration shows that before RNA processing, there is a primary RNA transcript including five boxes labeled, left to right, as exon 1, intron, exon 2, intron, and exon 3. After RNA processing, there is a spliced RNA with these parts, left to right: a 5' cap, a 5' untranslated region, exon 1, exon 2, exon 3, a 3' untranslated region, and a poly-a tail.

Figure 15.11 Eukaryotic mRNA contains introns that must be spliced out. A 5' cap and 3' poly-A tail are also added.

A scientist splices a eukaryotic promoter in front of a bacterial gene and inserts the gene in a bacterial chromosome. Would you expect the bacterium to transcribe the gene?

Initially the bacterium will not transcribe it, but will transcribe after 5’ capping.
It will not transcribe it because there is no poly A tail in prokaryotes.
No, prokaryotes use different promoters than eukaryotes.
Yes, the bacterium would transcribe the eukaryotic gene.

The mouse genome includes one gene and two pseudogenes for cytoplasmic thymidine kinase. Pseudogenes are genes that have lost their protein-coding ability or are no longer expressed by the cell. These pseudogenes are copied from mRNA and incorporated into the chromosome. For example, the mouse thymidine kinase promoter also has a conserved CAAT box (GGCCAATCT) at approximately -80. This sequence is essential and is involved in binding transcription factors. Further upstream of the TATA box, eukaryotic promoters may also contain one or more GC-rich boxes (GGCG) or octamer boxes (ATTTGCAT). These elements bind cellular factors that increase the efficiency of transcription initiation and are often identified in more “active” genes that are constantly being expressed by the cell.

Transcription Factors for RNA Polymerase II

The complexity of eukaryotic transcription does not end with the polymerases and promoters. An army of basal transcription factors, enhancers, and silencers also help to regulate the frequency with which pre-mRNA is synthesized from a gene. Enhancers and silencers affect the efficiency of transcription but are not necessary for transcription to proceed. Basal transcription factors are crucial in the formation of a preinitiation complex on the DNA template that subsequently recruits RNA polymerase II for transcription initiation.

The names of the basal transcription factors begin with “TFII” (this is the transcription factor for RNA polymerase II) and are specified with the letters A–J. The transcription factors systematically fall into place on the DNA template, with each one further stabilizing the preinitiation complex and contributing to the recruitment of RNA polymerase II.

The processes of bringing RNA polymerases I and III to the DNA template involve slightly less complex collections of transcription factors, but the general theme is the same. Eukaryotic transcription is a tightly regulated process that requires a variety of proteins to interact with each other and with the DNA strand. Although the process of transcription in eukaryotes involves a greater metabolic investment than in prokaryotes, it ensures that the cell transcribes precisely the pre-mRNAs that it needs for protein synthesis.

Everyday Connection for AP® Courses

During human embryonic development, a transcription factor encoded by the SRY gene starts a chain of events, causing the embryo to develop male sex characteristics. This gene is on the Y chromosome in humans and many other mammals. A deletion or mutation of the SRY gene can cause the human embryo to not develop into a male even though the individual has an XY genotype, a condition called Swyer syndrome.

A flow chart illustrates the steps in human embryonic development leading to normal male development. First the SRY gene of the Y chromosome in embryonic germ cells produces testis-determining SRY protein, which in turn initiates the production of multiple proteins that cause the gonad medulla to differentiate into a testis. This medulla contains Sertoli cells that secrete testosterone, which controls development of the Wolffian duct into accessory structures, and male external genitalia. The medulla also contains Leydig cells that secrete anti-Müllerian hormone, that causes the regression of the Müllerian duct, which would otherwise develop into female internal organs.

Figure 15.12 The SYR gene of the Y chromosome produces proteins that lead to the expression of primary sex characteristics, as shown.

The protein product of the SRY gene is a DNA binding protein. Together with a protein called SF1, the SRY protein acts as a transcription factor that “turns on” certain genes. Which of the following statements best describes how a change in these two proteins would affect male sexual development?

A mutation that abolished activity of SF1 would increase the effect of a SRY mutation, making the person more feminine.
A mutation that abolished activity of SF1 would cancel out a mutation in SRY, so if both mutations occur together male sex characteristics would develop normally.
A mutation in the SRY protein that abolished activity would result in abnormal development of male sex characteristics but a mutation of SF1 would not.
Both a mutation in the SRY protein and a mutation in SF1 that abolished activity would result in a lack of development of male sex characteristics.

Evolution Connection

The Evolution of Promoters

The evolution of genes may be a familiar concept. Mutations can occur in genes during DNA replication, and the result may or may not be beneficial to the cell. By altering an enzyme, structural protein, or some other factor, the process of mutation can transform functions or physical features. However, eukaryotic promoters and other gene regulatory sequences may evolve as well. For instance, consider a gene that, over many generations, becomes more valuable to the cell. Maybe the gene encodes a structural protein that the cell needs to synthesize in abundance for a certain function. If this is the case, it would be beneficial to the cell for that gene’s promoter to recruit transcription factors more efficiently and increase gene expression.

Scientists examining the evolution of promoter sequences have reported varying results. In part, this is because it is difficult to infer exactly where a eukaryotic promoter begins and ends. Some promoters occur within genes; others are located very far upstream, or even downstream, of the genes they are regulating. However, when researchers limited their examination to human core promoter sequences that were defined experimentally as sequences that bind the preinitiation complex, they found that promoters evolve even faster than protein-coding genes.

It is still unclear how promoter evolution might correspond to the evolution of humans or other higher organisms. However, the evolution of a promoter to effectively make more or less of a given gene product is an intriguing alternative to the evolution of the genes themselves.¹

According to this passage, which of the following has been shown to evolve faster than protein-coding genes?

core promoters that bind the preinitiation complex
core promoters that occur within genes
promoters that occur far upstream of the gene
promoters that occur downstream of a gene

Promoter Structures for RNA Polymerases I and III

In eukaryotes, the conserved promoter elements differ for genes transcribed by RNA polymerases I, II, and III. RNA polymerase I transcribes genes that have two GC-rich promoter sequences in the -45 to +20 region. These sequences alone are sufficient for transcription initiation to occur, but promoters with additional sequences in the region from -180 to -105 upstream of the initiation site will further enhance initiation. Genes that are transcribed by RNA polymerase III have upstream promoters or promoters that occur within the genes themselves.

Eukaryotic Elongation and Termination

Following the formation of the preinitiation complex, the polymerase is released from the other transcription factors, and elongation is allowed to proceed as it does in prokaryotes with the polymerase synthesizing pre-mRNA in the 5' to 3' direction. As discussed previously, RNA polymerase II transcribes the major share of eukaryotic genes, so this section will focus on how this polymerase accomplishes elongation and termination.

Although the enzymatic process of elongation is essentially the same in eukaryotes and prokaryotes, the DNA template is more complex. When eukaryotic cells are not dividing, their genes exist as a diffuse mass of DNA and proteins called chromatin. The DNA is tightly packaged around charged histone proteins at repeated intervals. These DNA–histone complexes, collectively called nucleosomes, are regularly spaced and include 146 nucleotides of DNA wound around eight histones like thread around a spool.

For polynucleotide synthesis to occur, the transcription machinery needs to move histones out of the way every time it encounters a nucleosome. This is accomplished by a special protein complex called FACT, which stands for “facilitates chromatin transcription.” This complex pulls histones away from the DNA template as the polymerase moves along it. Once the pre-mRNA is synthesized, the FACT complex replaces the histones to recreate the nucleosomes.

The termination of transcription is different for the different polymerases. Unlike in prokaryotes, elongation by RNA polymerase II in eukaryotes takes place 1,000–2,000 nucleotides beyond the end of the gene being transcribed. This pre-mRNA tail is subsequently removed by cleavage during mRNA processing. On the other hand, RNA polymerases I and III require termination signals. Genes transcribed by RNA polymerase I contain a specific 18-nucleotide sequence that is recognized by a termination protein. The process of termination in RNA polymerase III involves an mRNA hairpin similar to rho-independent termination of transcription in prokaryotes.

Footnotes

1H Liang et al., “Fast evolution of core promoters in primate genomes,” Molecular Biology and Evolution 25 (2008): 1239–44.

15.3 Eukaryotic Transcription