5¿ • Eukaryotic promoters are more varied than bacterial
promoters. Not only are different types of promoters
RNA Hairpin loop
(GC-rich)
UUUUUU 3¿
A A A A
U UU U A A
3¿ U 5¿
U
DNA
5¿ 3¿
Self-
RNA
complementary
sequence
polymerase
Figure 18-15 Termination of Transcription in Bacterial
Genes That Do Not Require the Rho Termination Factor. A
short self-complementary sequence near the end of the gene
allows the newly formed RNA molecule to form a hairpin loop struc-
ture that helps dissociate the RNA from the DNA template.
In contrast, RNA molecules that do not form a GC-rich
hairpin loop require participation of the rho factor for termi-
nation. Genes encoding such RNAs were first discovered in
experiments in which purified DNA obtained from bacterio-
phage l was transcribed with purified RNA polymerase. Some
genes were found to be transcribed into RNA molecules that
are longer than the RNAs produced in living cells, suggesting
that transcription was not terminating properly. This problem
could be corrected by adding rho factor, which binds to spe-
cific termination sequences 50–90 bases long located near the
3′ end of newly forming RNA molecules. The rho factor acts
as an ATP-dependent unwinding enzyme, moving along the
newly forming RNA molecule toward its 3′ end and unwind-
ing it from the DNA template as it proceeds.
Whether termination depends on rho or on the formation
of a hairpin loop, it results in the release of the newly tran-
scribed RNA molecule and the core RNA polymerase. The core
polymerase can then bind sigma factor again and reinitiate
RNA synthesis at another promoter.
Transcription in Eukaryotic Cells Has
Additional Complexity Compared with
Prokaryotes
Transcription in eukaryotic cells involves the same four stages
described in Figure 18-11, but the process in eukaryotes is
more complicated than that in bacteria. The main differences
are as follows:
• Three different RNA polymerases transcribe the nuclear
DNA of eukaryotes. Each synthesizes one or more classes
of RNA.
540
M18_HARD7694_09_GE_C18.indd 540
employed for the three polymerases, but there is great
variation within each type—especially among the ones
for protein-coding genes. Furthermore, some eukaryotic
promoters are actually located downstream from the tran-
scription start site.
• Binding of eukaryotic RNA polymerases to DNA requires
the participation of additional proteins, called transcrip-
tion factors. Unlike the bacterial sigma factor, eukaryotic
transcription factors are not part of the RNA polymerase
molecule itself. Rather, some of them must bind to DNA
before RNA polymerase can bind to the promoter and
initiate transcription. Thus, transcription factors, rather
than RNA polymerase itself, determine the specificity
of transcription in eukaryotes. In this chapter, we limit
our discussion to the class of factors that are essential
for the transcription of all genes transcribed by an RNA
polymerase. (We defer discussion of the regulatory class
of transcription factors, which selectively act on specific
genes, until Chapter 20.)
• Protein-protein interactions play a prominent role in the
first stage of eukaryotic transcription. Although some
transcription factors bind directly to DNA, many attach
to other proteins—either to other transcription factors or
to RNA polymerase itself.
• RNA cleavage is more important than the site where tran-
scription is terminated in determining the location of the
3′ end of the RNA product.
• Newly forming eukaryotic RNA molecules typi-
cally undergo extensive RNA processing (chemical
modification) both during and, to a larger extent, after
transcription.
We will now examine these various aspects of eukaryotic
transcription, starting with the existence of multiple forms of
RNA polymerase.
RNA Polymerases I, II, and III Carry Out
Transcription in the Eukaryotic Nucleus
Table 18-1 summarizes some properties of the three RNA
polymerases that function in the nucleus of the eukaryotic
cell, along with two other polymerases found in mitochondria
and chloroplasts. The nuclear enzymes are designated RNA
polymerases I, II, and III. As the table indicates, these enzymes
differ in their location within the nucleus and in the kinds of
RNA they synthesize. The nuclear RNA polymerases also dif-
fer in their sensitivity to various inhibitors, such as a-amanitin,
a deadly toxin produced by the mushroom Amanita phalloides
(the “death cap” fungus; the F-actin binding drug phalloidin,
introduced in Chapter 13, also comes from this organism).
RNA polymerase I resides in the nucleolus and is re-
sponsible for synthesizing an RNA molecule that serves as a
precursor for three of the four types of rRNA found in eukary-
otic ribosomes (28S rRNA, 18S rRNA, and 5.8S rRNA). This
enzyme is not sensitive to a-amanitin. Its association with the
nucleolus is understandable because the nucleolus is the site
of ribosomal RNA synthesis and ribosomal subunit assembly.
17/02/17 7:24 am
 Table 18-1 Properties of Eukaryotic RNA Polymerases
RNA Polymerase Location Main Products
I Nucleolus Precursor for 28S rRNA, 18S rRNA, and 5.8S rRNA
II Nucleoplasm Pre-mRNA, most snRNA, and microRNA
III Nucleoplasm Pre-tRNA, 5S rRNA, and other small RNAs
Mitochondrial Mitochondrion Mitochondrial RNA
Chloroplast Chloroplast Chloroplast RNA
*In mammals.
RNA polymerase II is found in the nucleoplasm and
synthesizes precursors to mRNA, the class of RNA molecules
that encode proteins. Rather than being diffusely distributed
throughout the nucleus, active molecules of polymerase II are
located in discrete clusters, called transcription factories, that
may represent sites where active genes come together to be
transcribed. In addition to producing mRNA precursors, RNA
polymerase II synthesizes most of the snRNAs, small nuclear
RNAs involved in posttranscriptional RNA processing, and the
microRNAs, which regulate the translation and stability of
specific mRNAs and, to a lesser extent, control the transcrip-
tion of certain genes. Polymerase II is responsible for produc-
ing the greatest variety of RNA molecules and is extremely
sensitive to a-amanitin, which explains the toxicity of this
compound to humans and other animals.
RNA polymerase II differs from polymerases I and III at its
C-terminus, where it has extra amino acids. The C-terminus
of RNA polymerase II can be phosphorylated at a variety of
locations to produce what is sometimes called a phosphoryla-
tion “code.” This “code” dramatically affects the functions of
polymerase II and correlates with where the enzyme is located
along the DNA as it continues transcription. As a result, this
most versatile of the RNA polymerases is also the most tightly
regulated.
RNA polymerase III is also a nucleoplasmic enzyme,
but it synthesizes a variety of small RNAs, including tRNA
precursors and the smallest type of ribosomal RNA, 5S rRNA.
Mammalian RNA polymerase III is sensitive to a-amanitin
but only at higher levels of the toxin than are required to in-
hibit RNA polymerase II. (The comparable enzymes of some
other eukaryotes, such as insects and yeasts, are insensitive
to a-amanitin.)
Structurally, RNA polymerases I, II, and III are somewhat
similar to each other as well as to bacterial core RNA poly-
merase. The three enzymes are all quite large, with multiple
polypeptide subunits and molecular weights around 500,000.
RNA polymerase II, for example, has more than ten subunits
of at least eight different types. The three biggest subunits are
evolutionarily related to the bacterial RNA polymerase sub-
units a, b, and b′. Three of the smaller subunits lack that
relationship but are also found in RNA polymerases II and
III. The RNA polymerases of mitochondria and chloroplasts
resemble their bacterial counterparts closely, as you might
expect from the probable origins of these organelles as en-
dosymbiotic bacteria (see Figure 4-16). Like bacterial RNA
polymerase, the mitochondrial and chloroplast enzymes are
resistant to a-amanitin.
M18_HARD7694_09_GE_C18.indd 541
A-Amanitin Sensitivity
Resistant
Very sensitive
Moderately sensitive*
Resistant
Resistant
Three Classes of Promoters Are Found in
Eukaryotic Nuclear Genes, One for Each Type
of RNA Polymerase
The promoters that eukaryotic RNA polymerases bind to are
even more varied than bacterial promoters, but they can be
grouped into three main categories, one for each type of poly-
merase. Figure 18-16 shows examples of the three types of
promoters.
The promoter used by RNA polymerase I—that is, the pro-
Chapter 18
moter of the transcription unit that produces the precursor for
the three largest rRNAs—has two parts (Figure 18-16a). The
part called the core promoter, defined as the smallest set of
DNA sequences able to direct the accurate initiation of tran-
|
scription by RNA polymerase, actually extends into the nucleo-
Gene Expression: I. The Genetic Code and Transcription
tide sequence to be transcribed. The core promoter is sufficient
for proper initiation of transcription, but transcription is made
more efficient by the presence of an upstream control element,
which for RNA polymerase I is a fairly long sequence similar
(though not identical) to the core promoter. Attachment of
transcription factors to both parts of the promoter facilitates
the binding of RNA polymerase I to the core promoter and en-
ables it to initiate transcription at the start site.
In the case of RNA polymerase II, at least four types of
DNA sequences are involved in core promoter function (Figure
18-16b). These four elements are (1) a short initiator (Inr)
sequence surrounding the transcriptional start site (which is
often an A, as in bacteria); (2) the TATA box, which consists of
a consensus sequence of TATA followed by two or three more
A’s, usually located about 25 nucleotides upstream from the
start site; (3) the TFIIB recognition element (BRE) located
slightly upstream of the TATA box; and (4) the downstream
promoter element (DPE) located about 30 nucleotides
downstream from the start site. These four elements are or-
ganized into two general types of core promoters: TATA-driven
promoters, which contain an Inr sequence and a TATA box
with or without an associated BRE, and DPE-driven promoters,
which contain DPE and Inr sequences but no TATA box or BRE.
Besides being found in eukaryotes, TATA-driven promoters are
also present in archaea, a key piece of evidence supporting the
idea that in some ways, archaea resemble eukaryotes more
closely than they resemble bacteria (see Table 4-1, page 102).
By itself, a core promoter (TATA-driven or DPE-driven) is
capable of supporting only a basal (low) level of transcription.
However, most protein-coding genes have additional short se-
quences further upstream—upstream control elements—that
improve the promoter’s efficiency. Some of these upstream
541
17/02/17 7:24 am
 Upstream control element Core promoter

Transcriptional start site

-180 -107 -45 +1 +20

DNA Transcription

(a) Promoter for RNA polymerase I

BRE TATA box Inr DPE

Transcriptional start site

Coding-strand sequences: (G/C)(G/C)(G/A)CGCC T A T A A A A Py2 C A Py5 G(A/T)CG

-25 +1

DNA Transcription

(b) Core promoter elements for RNA polymerase II

Promoter for tRNA gene

Transcriptional start site

+1 Box A Box B

DNA Transcription

Promoter for 5S-rRNA gene

Transcriptional start site

+1 Box A Box C

DNA Transcription

(c) Two types of promoters for RNA polymerase III

Figure 18-16 Examples of Eukaryotic Promoters for RNA Polymerases I, II, and III. (a) The promoter
for RNA polymerase I has two parts, a core promoter surrounding the start site and an upstream control ele-
ment. After the binding of appropriate transcription factors to both parts, the RNA polymerase binds to the core
promoter. (b) The typical promoter for RNA polymerase II has a short initiator (Inr) sequence, consisting mostly
of pyrimidines (Py), combined with either a TATA box or a downstream promoter element (DPE). Promoters
containing a TATA box may also include a TFIIB recognition element (BRE) as part of the core promoter. (c)
The promoters for RNA polymerase III vary in structure, but the ones for tRNA genes and 5S-rRNA genes are
located entirely downstream of the start site, within the transcribed sequence. Boxes A, B, and C are DNA
consensus sequences, each about 10 bp long. In tRNA genes, about 30–60 bp of DNA separate boxes A and B.
In 5S-rRNA genes, about 10–30 bp separate boxes A and C.

elements are common to many different genes; examples
include the CAAT box (consensus sequence GCCCAATCT
in animals and yeasts) and the GC box (consensus sequence
GGGCGG). The locations of these elements relative to a gene’s
transcriptional start site vary from gene to gene. The elements
within 100–200 nucleotides of the start site are often called
proximal control elements to distinguish them from enhancer
elements, which tend to be farther away and can even be lo-
cated downstream of the gene. When activating proteins bind
to enhancers, they locally change the conformation of DNA
near promoters by allowing chromatin remodeling proteins
542
and histone modifying enzymes to alter chromatin structure
and by promoting assembly of the basal transcriptional ma-
chinery. Keep in mind that for genes transcribed using RNA
polymerase II, these long-range elements are often crucial
for determining whether a gene is “switched on” (that is, it is
transcribed efficiently) or not (you will learn more about prox-
imal control elements and enhancers in Chapter 20).
The sequences important in promoter activity are of-
ten identified by deleting specific sequences from a cloned
DNA molecule, which is then tested for its ability to serve as
a template for gene transcription, either in a test tube or after

M18_HARD7694_09_GE_C18.indd 542 17/02/17 7:24 am

 introduction of the DNA into cultured cells. For example, Core promoter
when transcription of the gene for b-globin (the b chain of Start
TATA
hemoglobin) is investigated in this way, deletion of either the DNA
TATA box or an upstream CAAT box reduces the rate of tran-
scription at least tenfold. 1 TFIID binds to TBP
In contrast to RNA polymerases I and II, the RNA poly- TATA box in DNA. TFIID
merase III molecule uses promoters that are entirely down-
stream of the transcription unit’s transcriptional start site
when transcribing genes for tRNAs and 5S rRNA. The pro-
moters used by tRNA and 5S-rRNA genes are different, but
in both cases the consensus sequences fall into two blocks of
TFIIA
about 10 bp each (Figure 18-16c). The tRNA promoter has TFIIB
consensus sequences called box A and box B. The promoters 2 TFIIA and TFIIB
form complex with
for 5S-rRNA genes have box A (positioned farther from the
TFIID.
start site than in tRNA-gene promoters) and another critical
sequence, called box C. (Not shown in the figure is a third
type of RNA polymerase III promoter, an upstream promoter
TFIIF RNA polymerase II
that is used for the synthesis of other kinds of small RNA
molecules.) C-terminal
domain
The promoters used by all the eukaryotic RNA polymer- 3 Resulting complex is
(CTD) “tail”
ases must be recognized and bound by transcription factors bound by RNA polymerase
attached to TFIIF.
before the RNA polymerase molecule can bind to DNA. We
turn now to these transcription factors.

Chapter 18
General Transcription Factors Are Involved in
the Transcription of All Nuclear Genes

|
A general transcription factor is a protein that is always

Gene Expression: I. The Genetic Code and Transcription

required for an RNA polymerase molecule to bind to its pro-
moter and initiate RNA synthesis, regardless of the identity of
the gene involved. Eukaryotes have many such transcription TFIIE
factors; their names usually include “TF” (for transcription 4 Preinitiation complex
factor), a roman numeral identifying the polymerase they aid, TFIIH is completed by addition
of TFIIE and TFIIH.
and a capital letter that identifies each individual factor (for
example, TFIIA, TFIIB).
Using RNA polymerase II as an example, Figure 18-17
illustrates the involvement of general transcription factors
in the binding of RNA polymerase to a TATA-containing pro-
moter site in DNA. General transcription factors bind to pro-
moters in a defined order, starting with TFIID. Notice that al-
though TFIID binds directly to a DNA sequence (the TATA box
ATP
in this example or the DPE sequence in the case of DPE-driven
promoters), the other transcription factors interact primarily
with each other. Hence, protein-protein interactions play a
crucial role in the binding stage of eukaryotic transcription.
RNA polymerase II does not bind to the DNA until several P
steps into the process. Eventually, a large complex of proteins, 5 RNA polymerase P P P P
CTD undergoes
including RNA polymerase, becomes bound to the promoter phosphorylation. Transcription begins
region to form a preinitiation complex.
Before RNA polymerase II can actually initiate RNA syn-
thesis, it must be released from the preinitiation complex. A Figure 18-17 Role of General Transcription Factors in
key role in this process is played by the general transcription Binding RNA Polymerase II to DNA. This figure outlines the
sequential binding of six general transcription factors (called
factor TFIIH, which possesses both a helicase activity that
TFII_, where _ is a letter identifying the particular factor) and RNA
unwinds DNA and a protein kinase activity that catalyzes
polymerase. After the final activation step involving ATP-dependent
the phosphorylation of RNA polymerase II. Phosphorylation
phosphorylation of the RNA polymerase, the polymerase can initiate
allows RNA polymerase to detach from the transcription fac- transcription. In intact chromatin, the efficient binding of general
tors so that it can initiate RNA synthesis at the transcriptional transcription factors and RNA polymerase to DNA requires the
start site. At the same time, the helicase activity of TFIIH is participation of additional regulatory proteins that open up chroma-
thought to unwind the DNA so that the RNA polymerase mol- tin structure and facilitate assembly of the preinitiation complex at
ecule can begin to move. specific genes (see Figure 20-24).
543

M18_HARD7694_09_GE_C18.indd 543 17/02/17 7:24 am

 The region of RNA polymerase II that is phosphorylated
by TFIIH is very important for regulating RNA polymerase
II function. The large subunit of RNA polymerase II has a
C-terminal domain (CTD) that functions as a “tail.” The CTD
is much longer than the core portion of the polymerase; it is
the region of the polymerase that is phosphorylated to allow
elongation of the RNA. The CTD contains a series of seven
repeated amino acids; in yeast there are 27 of these repeats,
whereas in humans there are 52. Each repeat contains sites
that can be phosphorylated by specific kinases, including the
kinase that is part of TFIIH.
TFIID, the initial transcription factor to bind to the pro-
moter, is worthy of special note. Its ability to recognize and
bind to DNA promoter sequences is conferred by one of its
subunits, the TATA-binding protein (TBP), which com-
bines with a variable number of additional protein subunits
to form TFIID. Despite its name, the ability of TBP to bind
to DNA, illustrated in Figure 18-18, is not restricted to TA-
TA-containing promoters. TBP can also bind to promoters
TBP saddles
DNA
(a) End view
TBP saddles
DNA
Kink in DNA
(b) Side view
Figure18-18 TATA-Binding Protein (TBP) Bound to DNA. In
this computer graphic model, human TBP (purple) is shown bound
to DNA (blue). TBP differs from most DNA-binding proteins in that
it interacts with the minor groove of DNA, rather than the major
groove, and imparts a sharp bend to the DNA. When TBP is bound
to DNA, other transcription factors can interact with the convex
surface of the TBP “saddle.” TBP is involved in transcription initiation
for all types of eukaryotic promoters.
544
M18_HARD7694_09_GE_C18.indd 544
lacking a TATA box, including promoters used by RNA poly-
merases I and III. Depending on the type of promoter, TBP
associates with different proteins, and for promoters lacking a
TATA box, much of TBP’s specificity is probably derived from
its interaction with these associated proteins. TBP binds the
minor groove of DNA, causing a severe kink in the DNA that
promotes the attachment of other components of the preini-
tiation complex.
In addition to general transcription factors and RNA
polymerase II, several other kinds of proteins are required for
the efficient transcription and regulated activation of specific
genes. Some of these proteins are involved in opening up chro-
matin structure to facilitate the binding of RNA polymerase to
DNA. Others are regulatory transcription factors, which activate
specific genes by binding to upstream control elements and
recruiting coactivator proteins that in turn facilitate assembly
of the RNA polymerase preinitiation complex. (The identi-
ties and roles of these additional proteins will be described in
Chapter 20, which covers the regulation of gene expression;
see Figure 20-24.)
Elongation, Termination, and RNA Cleavage
Are Involved in Completing Eukaryotic RNA
Synthesis
After initiating transcription, RNA polymerases move along
the DNA and synthesize a complementary RNA copy of the
DNA template strand. Special proteins facilitate the disassem-
bly of nucleosomes in front of the moving polymerase and
their immediate reassembly after the enzyme passes. If an area
of DNA damage is encountered, RNA polymerase may become
stalled temporarily while the damage is corrected by proteins
that carry out DNA excision repair (see Figure 17-26).
Termination of transcription is governed by an assort-
ment of signals that differ for each type of RNA polymerase.
For example, transcription by RNA polymerase I is terminated
by a protein factor that recognizes an 18-nucleotide termina-
tion signal in the growing RNA chain. Termination signals
for RNA polymerase III are also known; they always include
a short run of U’s (as in bacterial termination signals), and
no ancillary protein factors are needed for their recognition.
Hairpin structures do not appear to be involved in termination
by either polymerase I or polymerase III.
For RNA polymerase II, transcripts destined to become
mRNA are often cleaved at a specific site before transcription
is actually terminated. The cleavage site is 10–35 nucleotides
downstream from a special AAUAAA sequence in the grow-
ing RNA chain. The polymerase may continue transcription
for hundreds or even thousands of nucleotides beyond the
cleavage site, but this additional RNA is quickly degraded. The
cleavage site is also the site for the addition of a poly(A) tail, a
string of adenine nucleotides found at the 3′ end of almost all
eukaryotic mRNAs. Addition of the poly(A) tail is part of RNA
processing, our next topic.
CONCEPT CHECK 18-2
Compare and contrast bacterial and eukaryotic transcription,
focusing on initiation and termination. What features are
similar? What features are different?
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