Proteins That Activate Transcription Work Through Protein ⬊Protein Contacts with RNA Polymerase Although transcriptional control is governed by a variety of mechanisms, an underly-ing p
Trang 1protein⬊protein interactions are an essential component of transcriptional activation.
We see this latter feature in the activation of RNA polymerase by CAP–(cAMP)2, for
example Third, the regulator proteins receive cues that signal the status of the
envi-ronment (for example, Trp, lactose, cAMP) and act to communicate this information
to the genome, typically via the medium of conformational changes and
DNA⬊pro-tein interactions
Proteins That Activate Transcription Work Through
Protein ⬊Protein Contacts with RNA Polymerase
Although transcriptional control is governed by a variety of mechanisms, an
underly-ing principle of transcriptional activation has emerged Transcriptional activation can
take place when a transcriptional activator protein [such as CAP–(cAMP)2] bound to
DNA makes protein⬊protein contacts with RNA polymerase, and the degree of
tran-scriptional activation is proportional to the strength of the protein:protein
interac-tion Generally speaking, a nucleotide sequence that provides a binding site for a
DNA-binding protein can serve as an activator site if the DNA-binding protein bound
there can interact with promoter-bound RNA polymerase These interactions can
involve either the -, -, -, or -subunits of RNA polymerase Moreover, if the
DNA-bound transcriptional activator makes contacts with two different components
of RNA polymerase, a synergistic effect takes place such that transcription is markedly
elevated Thus, transcriptional activation at specific genes relies on the presence of
one or more activator sites where one or more transcriptional activator proteins can
bind and make contacts with RNA polymerase bound at the promoter of the gene
In-deed, transcriptional activators may facilitate the recruitment and binding of RNA
polymerase to the promoter This general principle applies to transcriptional
activa-tion in both prokaryotic and eukaryotic cells In eukaryotes, transcripactiva-tional activators
typically have discrete domains of protein structure dedicated to DNA binding (DB
domains) and transcriptional activation (TA domains)
DNA Looping Allows Multiple DNA-Binding Proteins
to Interact with One Another
Because transcription must respond to a variety of regulatory signals, multiple
pro-teins are essential for appropriate regulation of gene expression These regulatory
proteins are the sensors of cellular circumstances, and they communicate this
infor-mation to the genome by binding at specific nucleotide sequences However, DNA is
virtually a one-dimensional polymer, and there is little space for a lot of proteins to
bind at (or even near) a transcription initiation site DNA looping permits additional
proteins to convene at the initiation site and to exert their influence on creating and
activating an RNA polymerase initiation complex (Figure 29.22) The number of
par-ticipants in transcriptional regulation is greatly expanded by DNA looping
Enhancer
Promoter TATA Initiator
RNA polymerase
Transcription
activator
FIGURE 29.22 Formation of a DNA loop deliv-ers DNA-bound transcriptional activator to RNA polymerase positioned at the promoter Protein ⬊protein interactions between the transcriptional activator and RNA polymerase activate transcription.
Trang 229.3 How Are Genes Transcribed in Eukaryotes?
Although the mechanism of transcription in prokaryotes and eukaryotes is funda-mentally similar, transcription is substantially more complicated in eukaryotes The significant difference is that the DNA of eukaryotes is wrapped around histones to form nucleosomes, and the nucleosomes are further organized into chromatin
(see Chapter 11) Nucleosomes repress gene expression Nucleosomes control gene
ex-pression by controlling access of the transcriptional apparatus to genes Two classes
of transcriptional co-regulators are necessary to overcome nucleosome repression: (1) enzymes that covalently modify the nucleosome histone proteins and thereby loosen histone⬊DNA interactions and (2) ATP-dependent chromatin-remodeling complexes However, gene activation depends not only on relief from nucleosome repression but also on interaction of RNA polymerase with the promoter Only those genes activated by specific positive regulatory mechanisms are transcribed A general understanding of transcription in eukaryotes rests on the following topics:
• The three classes of RNA polymerase in eukaryotes: RNA polymerases I, II, and III
• The structure and function of RNA polymerase II, the mRNA-synthesizing RNA polymerase
• Transcription regulation in eukaryotes, including:
– General features of gene regulatory sequences: promoters, enhancers, and re-sponse elements
– Transcription initiation by RNA polymerase II The general transcription factors (GTFs) Alleviating the repression due to nucleosomes Histone acetyl transferases (HATs)
Chromatin-remodeling complexes
• A general model for eukaryotic gene activation, based on the preceding
We turn now to a review of these various features of eukaryotic transcription
Eukaryotes Have Three Classes of RNA Polymerases
Eukaryotic cells have three classes of RNA polymerase, each of which synthesizes a
different class of RNA All three enzymes are found in the nucleus RNA polymerase
I is localized to the nucleolus and transcribes the major ribosomal RNA genes RNA
polymerase IItranscribes protein-encoding genes, and thus it is responsible for the
synthesis of mRNA RNA polymerase III transcribes tRNA genes, the ribosomal RNA
genes encoding 5S rRNA, and a variety of other small RNAs, including several in-volved in mRNA processing and protein transport
All three RNA polymerase types are large, complex multimeric proteins (500 to
700 kD), consisting of ten or more types of subunits Although the three differ in over-all subunit composition, they have several smover-aller subunits in common Furthermore, all possess two large subunits (each 140 kD or greater) having sequence similarity to the large - and -subunits of E coli RNA polymerase, indicating that the
funda-mental catalytic site of RNA polymerase is conserved among its various forms
In addition to their different functions, the three classes of RNA polymerase can be distinguished by their sensitivity to ␣-amanitin (Figure 29.23), a bicyclic octapeptide
produced by the poisonous mushroom Amanita phalloides (the “destroying angel”
resistant to this compound, RNA polymerase II is very sensitive and RNA polymerase III is less sensitive
The existence of three classes of RNA polymerases acting on three distinct sets
of genes implies that at least three categories of promoters exist to maintain this specificity Eukaryotic promoters are very different from prokaryotic promoters All three eukaryotic RNA polymerases interact with their promoters via so-called
transcription factors—DNA-binding proteins that recognize and accurately initiate transcription at specific promoter sequences For RNA polymerase I, its templates
CH
C
H
CH 2
C N CH N
H 3 C
H 2 C S O
O OH O
O
CH 2 C O C
CH CH
O
C O
C O
CH
CH 3
CH 3
CH 2 OH
HN
OH
HC C N
NH
NH
C O
C
H N
H
C
CH 2 CONH 2
N
H
HO
-Amanitin
FIGURE 29.23 The structure of -amanitin, one of a
series of toxic compounds known as amatoxins that are
found in the mushroom Amanita phalloides.
Trang 3are the rRNA genes Ribosomal RNA genes are present in multiple copies Optimal
expression of these genes requires the first 150 nucleotides in the immediate
5-upstream region
RNA polymerase III interacts with transcription factors TFIIIA, TFIIIB, and
TFIIIC.Interestingly, TFIIIA and/or TFIIIC bind to specific recognition sequences
that in some instances are located within the coding regions of the genes, not in the
5-untranscribed region upstream from the transcription start site TFIIIB associates
with TFIIIA or TFIIIC already bound to the DNA RNA polymerase III then binds
to TFIIIB to establish an initiation complex
RNA Polymerase II Transcribes Protein-Coding Genes
As the enzyme responsible for the regulated synthesis of mRNA, RNA polymerase II
has aroused greater interest than RNA polymerases I and III RNA polymerase II must
be capable of transcribing a great diversity of genes, yet it must carry out its function
at any moment only on those genes whose products are appropriate to the needs of
the cell in its ever-changing metabolism and growth The RNA polymerase II from
yeast (Saccharomyces cerevisiae) has been extensively characterized, and its structure has
been solved by x-ray crystallography (Figure 29.24) Strong homology between yeast
and human RNA polymerase II subunits suggests that the yeast RNA polymerase II is
an excellent model for human RNA polymerase II The yeast RNA polymerase II
consists of 12 different polypeptides, designated RPB1 through RPB12 and ranging
in size from 192 to 8 kD (Table 29.2) RPB3, RPB4, and RPB7 are unique to RNA
polymerase II, whereas RPB5, RPB6, RPB8, and RPB10 are common to all three
eukaryotic RNA polymerases
RNA polymerases adopt a clawlike structure, grasping the DNA duplex as shown
in Figure 29.24 for yeast RNA polymerase II The DNA strands are unwound and
separated, and the template strand enters the active site, where template-directed
NTP substrate selection and NMP addition to the growing RNA transcript occur
NTP substrates access the active site through a channel in the floor The DNA:RNA
hybrid duplex exits at a right angle from the active site As the hybrid duplex
emerges from the top of the protein structure, the RNA transcript is separated from
the DNA template by RPB1 residue Phe252, which acts as a wedge, splitting the
po-sition10 RNA:DNA base pair (the base pair located 10 bases from the active site)
The template strand is now free to re-anneal with the nontemplate strand to
re-establish the dsDNA structure
FIGURE 29.24 Structure of the yeast RNA polymerase (pdb id 1Y77).The template DNA strand is shown in green, the nontemplate DNA strand in blue The RNA transcript (hot pink) is emerging at the bottom of the structure RPB1, the largest polypeptide chain, is shown
in orange, its C-terminal domain (CTD) is to the upper right The active-site Mg2is shown as a red sphere The atoms of a ribonucleotide substrate analog (GMPCP) are shown as dark blue spheres.
Trang 4The RPB1 subunit has an unusual structural feature not found in prokaryotes: Its
C-terminal domain (CTD)contains 27 repeats of the amino acid sequence YSPTSPS (The analogous subunit in RNA polymerase II enzymes of other eukaryotes has this heptapeptide tandemly repeated as many as 52 times.) Note that the side chains of
5 of the 7 residues in this repeat have OOH groups, endowing the CTD with
con-siderable hydrophilicity and multiple sites for phosphorylation A number of CTD
kinases have been described, targeting different residues at different stages of the transcription process The CTD domain may project more than 50 nm from the sur-face of RNA polymerase II
The CTD is essential to RNA polymerase II function Only RNA polymerase II whose CTD is not phosphorylated can initiate transcription However, transcription elongation proceeds only after protein phosphorylation within the CTD, suggesting that phosphorylation triggers the conversion of an initiation complex into an elon-gation complex Such a mechanism would allow protein phosphorylation to regu-late gene expression Following termination of transcription, a phosphatase recycles RNA polymerase II to its unphosphorylated form The CTD also plays a prominent role in orchestrating subsequent events in the transcription process A multitude of additional proteins are essential to the formation of a translatable mRNA from the primary RNA polymerase II transcript; these proteins (described later in this chap-ter) include 5-capping enzymes, splicing factors, and 3-polyadenylylation com-plexes Recruitment of these proteins is dependent upon phosphorylation of Ser residues at positions 2 and 5 in the CTD heptapeptide repeat Phosphorylation of these Ser residues is also a prerequisite for interactions between the CTD and the histone methyltransferases capable of remodeling nucleosomes into a transcrip-tionally permissive state
The Regulation of Gene Expression Is More Complex in Eukaryotes
Not only metabolic activity and cell division but also complex patterns of embryonic development and cell differentiation must be coordinated through the regulation of gene expression All this coordinated regulation takes place in cells where the relative quantity (and diversity) of DNA is very great: A typical mammalian cell has 1500 times
as much DNA as an E coli cell The structural genes of eukaryotes are rarely organized
in clusters akin to operons Each eukaryotic gene typically possesses a discrete set of regulatory sequences appropriate to the requirements for regulating its transcription Certain of these sequences provide sites of interaction for general transcription factors,
RPB5 25 In polymerases I, II, and III RPB6 18 In polymerases I, II, and III
RPB8 17 In polymerases I, II, and III
RPB10 8 In polymerases I, II, and III
RPB12 8 In polymerases I, II, and III
*A very similar RNA polymerase II can be isolated from human cells.
†RPB stands for RNA polymerase B; RNA polymerases I, II, and III are sometimes called RPA, RPB, and RPC.
Adapted from Myer, V E., and Young, R A., 1998 RNA polymerase II holoenzymes and subcomplexes The Journal of
Biologi-cal Chemistry 273:27757–27760.
TABLE 29.2 Yeast* RNA Polymerase II Subunits
Trang 5whereas others endow the gene with great specificity in expression by providing targets
for specific transcription factors
Gene Regulatory Sequences in Eukaryotes Include Promoters,
Enhancers, and Response Elements
RNA polymerase II promoters commonly consist of two separate sequence features:
the core element, near the transcription start site, where general transcription factors
(GTFs) bind, and more distantly located regulatory elements, known variously as
enhancers or silencers These regulatory sequences are recognized by specific
DNA-binding proteins that activate transcription above basal levels (enhancers bind
transcriptional activators) or repress transcription (silencers bind repressors) The site of
transcription initiation, called initiator (Inr), has the consensus sequence (Py)2CA(Py)5
(two pyrimidines, then CA, then five pyrimidines) located between positions 3 and
5, where 1 is the transcription start site The core region often consists of a TATA
box(a TATAAA consensus element) indicating the transcription start site; the TATA
motif is usually located at position 25 (Figure 29.25) Genes that lack a TATA often
have a downstream promoter element (DPE) centered on the 30 region Other
reg-ulatory elements include short nucleotide sequences (sometimes called response
elements) found near the promoter (the promoter-proximal region) that can bind certain
specific transcription factors, such as proteins that trigger expression of a related set of
genes in response to some physiological signal (hormone) or challenge (temperature
shock)
Promoters The promoters of eukaryotic genes encoding proteins can be quite
com-plex and variable, but they typically contain modules of short conserved sequences,
such as the TATA box, the CAAT box, and the GC box Sets of such modules
embed-ded in the upstream region collectively define the promoter The presence of a CAAT
box, usually located around 80 relative to the transcription start site, signifies a strong
promoter One or more copies of the sequence GGGCGG or its complement (referred
to as the GC box) have been found upstream from the transcription start sites of
“housekeeping genes.” Housekeeping genes encode proteins commonly present in all
cells and essential to normal function; such genes are typically transcribed at more or
less steady levels Figure 29.26 depicts the promoter regions of several representative
eukaryotic genes Table 29.3 lists transcription factors that bind to respective modules
These transcription factors typically behave as positive regulatory proteins essential to
transcriptional activation by RNA polymerase II at these promoters
Enhancers Eukaryotic genes have, in addition to promoters, regulatory sequences
known as enhancers Enhancers (also called upstream activation sequences, or UASs)
assist initiation Enhancers differ from promoters in two fundamental ways First, the
location of enhancers relative to the transcription start site is not fixed Enhancers may
A
Inr
A
A
A
Chicken
ovalbumin
Adenovirus
late
Rabbit
-globin
T
8 2
T
3 7
T
3 7
A
9 7 8 5A 6 3A 5 0A
+1 Transcription start site A
8 3 T
9 3
Mouse-globin
major
A
A
A
G
A
T
A
G
A
T
G
C
G
C
G
A
G
C
T
G
G
C
G
A
G
C
A
G
G
A
G
C
T
G
G
A
G
G
T
G
G
G
A
G
G
C
G
G
G
T
G
C
G
C
A
A
C
A
T
G
G
G
C
C
C
C
A
T
G
C
G
G
T
A
T
C
T
G
T
T
C
T
G
G
G
G
C
C
T
T
T
T
C
C
C
G
C
T
C
C
T
G
T
T
C
T
C
A
C
C
C
C
T
A
A
A
G
A
G
G
G
G
G
G
G
A
C
G
C
G
A
C
T
C
T
T
A
A
A
A
T
T
A
T
A
A
A
A
T
T
A
G
G
T
T
C
T
C
T
T T
FIGURE 29.25 The Inr and TATA box in selected eukary-otic genes The consensus sequence of a number of such promoters is presented in the lower part of the fig-ure, the numbers giving the percent occurrence of vari-ous bases at the positions indicated.
Trang 6be several thousand nucleotides away from the promoter, and they act to enhance
tran-scription initiation even if positioned downstream from the gene Second, enhancer se-quences are bidirectional in that they function in either orientation That is, enhancers
can be removed and then reinserted in the reverse sequence orientation without im-pairing their function Like promoters, enhancers represent modules of consensus se-quence Enhancers are “promiscuous,” because they stimulate transcription from any
promoter that happens to be in their vicinity Nevertheless, enhancer function is
depen-dent on recognition by a specific transcription factor A specific transcription factor bound at
an enhancer element stimulates transcription by interacting with RNA polymerase II
at a nearby promoter
Response Elements Promoter modules in genes responsive to common regulation
are termed response elements Examples include the heat shock element (HSE), the
glucocorticoid response element (GRE), and the metal response element (MRE).
These various elements are found in the promoter regions of genes whose
transcrip-SV40 early region
(a)
(b)
Histone H2B
Thymidine kinase
Thymidine kinase
–140 –120 –100
Octamer CAAT GC TATA Initiator
–80 –60 –40 –20
mRNA
mRNA
Controls initial binding of RNA polymerase
Controls choice
of transcription startpoint
FIGURE 29.26 Promoter regions of several
representa-tive eukaryotic genes (a) The SV40 early genes, the
his-tone H2B gene, and the thymidine kinase gene Note
that these promoters contain different combinations of
the various modules In (b), the function of the modules
within the thymidine kinase gene is shown.
Sequence Consensus
Adapted from Lewin, B., 1994 Genes V Cambridge, MA: Cell Press.
TABLE 29.3 A Selection of Consensus Sequences That Define Various RNA Polymerase II Promoter
Modules and the Transcription Factors That Bind to Them
Trang 7tion is activated in response to a sudden increase in temperature (heat shock),
glu-cocorticoid hormones, or toxic heavy metals, respectively (Table 29.4) HSE
se-quences are recognized by a specific transcription factor, HSTF (for heat shock
transcription factor) HSEs are located about 15 bp upstream from the transcription
start site of a variety of genes whose expression is dramatically enhanced in response
to elevated temperature Similarly, the response to steroid hormones depends on the
presence of a GRE positioned 250 bp upstream of the transcription start point
Acti-vation of the steroid receptor (a specific transcription factor) at a GRE occurs when
certain steroids bind to the steroid receptor
Many genes are subject to multiple regulatory influences Regulation of such
genes is achieved through the presence of an array of different regulatory
ele-ments The metallothionein gene is a good example (Figure 29.27)
Metallo-thionein is a metal-binding protein that protects cells against metal toxicity by
binding excess amounts of heavy metals and removing them from the cell This
protein is always present at low levels, but its concentration increases in response
to heavy metal ions such as cadmium or in response to glucocorticoid hormones
The metallothionein gene promoter consists of two general promoter elements,
namely, a TATA box and a GC box, two basal-level enhancers, four MREs, and one
GRE These elements function independently of one another; any one is able to
activate transcription of the gene
Transcription Initiation by RNA Polymerase II Requires TBP
and the GTFs
A eukaryotic transcription initiation complex consists of RNA polymerase II, five
general transcription factors (GTFs), and a 20-subunit complex called Mediator (or
Srb/Med) The CTD of RNA polymerase II anchors Mediator to the polymerase
Mediator allows RNA polymerase II to communicate with transcriptional activators
Physiological Response
Adapted from Lewin, B., 1994 Genes V Cambridge, MA: Cell Press.
TABLE 29.4 Response Elements That Identify Genes Coordinately Regulated in Response to Particular Physiological Challenges
Response
elements
Protein
binding
mRNA
ANIMATED FIGURE 29.27 The metallothionein gene possesses several constitutive elements in
its promoter (the TATA and GC boxes) as well as specific response elements such as MREs and a GRE The BLEs are
elements involved in basal level expression (constitutive expression) TRE is a tumor response element activated
in the presence of tumor-promoting phorbol esters such as TPA (tetradecanoyl phorbol acetate) See this figure
animated at www.cengage.com/login.
Trang 8bound at sites distal from the promoter There are six GTFs (Table 29.5), five of which
are required for transcription: TFIIB, TFIID, TFIIE, TFIIF, and TFIIH The sixth,
TFIIA,stimulates transcription by stabilizing the interaction of TFIID with the TATA
box TFIID consists of TBP (TATA-binding protein), which directly recognizes the TATA box within the core promoter, and a set of TBP-associated factors (TAFs or
TAF II s).5Some TAFs resemble histones and may form a histone octamerlike structure that facilitates TFIID:DNA interactions The TBP–TAFIIcomplexes serve as a bridge between the promoter and RNA polymerase II Some are capable of recognizing core promoters lacking a TATA box TBP binds to the core promoter through contacts made with the minor groove of the DNA, distorting and bending the DNA so that DNA sequences upstream and downstream of the TATA box come into closer proximity (Figure 29.28a) Once TBP/TFIID is bound at the core promoter, a complex contain-ing RNA polymerase II and the remaincontain-ing GTFs convenes at this site, establishcontain-ing a
competent transcription preinitiation complex (Figure 29.28b) An open complex then
forms, and transcription begins
The Role of Mediator in Transcription Activation and Repression
Transcription activation requires Mediator Mediator serves as a bridge between
gene-specific transcription co-activators bound to enhancers and the RNA polymerase II/GTF transcription machinery bound at the promoter Once DNA becomes accessi-ble through the action of chromatin remodeling complexes (discussed in the following sections), a transcription co-activator binds to an enhancer and recruits Mediator to the gene Mediator then establishes the bridge by promoting the binding of GTFs and RNA polymerase II at the promoter Mediator has been described as the ultimate regulator
of transcription since it integrates and communicates information from enhancers and transcription co-activators to RNA polymerase at the promoter
The Mediator complex is about 1 megadalton in mass, with a core structure
com-posed of about 20 distinct subunits (in yeast; about 30 in humans), the MED proteins.
Mediator is a somewhat crescent-shaped structure, with a head, middle, and tail Eight MED proteins each are found in the head and middle regions, and four make up the tail The tail section recognizes and binds the transcription co-activator Both the head region and the middle region of Mediator bind RNA polymerase II and interact with its CTD, with the middle region also associating with general transcription factor TFIIE (Figure 29.29a)
Two of the Mediator subunits are a cyclin-CDK pair (CycC/CDK8), and they act
to phosphorylate S5 in the CTD YSPTSPS heptapeptide repeat Mediator is a global
Number of Factor Subunits Function
TFIID
TBP 1 Core promoter recognition (TATA); TFIIB recruitment
TAFs 14 Core promoter recognition (non-TATA elements); positive and negative regulatory functions; HAT
(histone acetyltransferase) activity TFIIA 3 Stabilization of TBP binding; stabilization of TAF–DNA interactions
TFIIB 1 RNA polymerase II-TFIIF recruitment; start-site selection by RNA polymerase II
TFIIF 2 Promoter targeting of polymerase II; destabilization of nonspecific RNA polymerase II–DNA interactions RNA pol II 12 Enzymatic synthesis of RNA; TFIIE recruitment
TFIIE 2 TFIIH recruitment; modulation of TFIIH helicase, ATPase, and kinase activities; promoter melting TFIIH 9 Promoter melting using helicase activity; promoter clearance via CTD phosphorylation (2 subunits of
TFHII are a cyclin⬊CDK pair)
Adapted from Table 1 in Roeder, R G., 1996 The role of general initiation factors in transcription by RNA polymerase II Trends in Biochemical Sciences 21:327–335; and Reese, J C., 2003 Basal transcription factors Current Opinion in Genetics and Development 13:114–118.
TABLE 29.5 General Transcription Initiation Factors from Human Cells
5 For many genes, another transcription factor called SAGA (which also contains TAF II s) can serve in-stead of TFIID to initiate transcription.
Trang 9regulator essential to transcription of virtually every RNA polymerase II-dependent
gene Mediator is even required for basal transcription of these genes Mediator
also displays HAT activity, which may aid in exposing promoters and subsequent
binding of GTFs and RNA polymerase II Taken together, the results indicate that
Mediator provides a scaffold for assembly of the pre-initiation complex (PIC), the
RNA pol II IID IID
Preinitiation complex
CTD
RNA pol II
(b)
(a)
II E
IIB
II F
II F
II F
II F IIH
IIB
II E
IIH
T AT A
TA T A
Inr
Inr
FIGURE 29.28 Transcription initiation (a) Model of the TATA-binding
protein (TBP) in complex with a DNA TATA sequence (pdb id 1YTB).
The saddle-shaped TBP (gold) is unusual in that it binds in the minor
groove of DNA, sitting on the DNA like a saddle on a horse TBP
binding pries open the minor groove, creating a 100° bend in the
DNA axis and unwinding the DNA within the TATA sequence The
other subunits of the TFIID complex (see Table 29.5) sit on TBP, like a
“cowboy on a saddle.” All known eukaryotic genes (those lacking a
TATA box as well as those transcribed by RNA polymerase I or III) rely
on TBP (b) Formation of a preinitiation complex at a TATA-containing
promoter TFIID bound to the TATA motif recruits RNA polymerase II
and the other GTFs to form the preinitiation complex Melting of the
DNA duplex around Inr generates the open complex and
transcrip-tion ensues.
middle
promoter
head
Mediator
tail
DNA enhancer
(a)
Co-Act
RNA pol II
II F
II D II B II EII H
silencer
CTD
II F
II B
II E
II D RNA
pol II
II H
promoter
Co-Rep
MED12 MED13 CycC CDK8
(b)
FIGURE 29.29 Simple models of Mediator in the regulation of eukaryotic gene transcription (a) Mediator as a
transcription activator Mediator regions are highlighted in color: green for the tail, yellow for the middle, and
red for the head RNA polymerase II and the GTFs are blue The transcription co-activator is orange DNA is
shown as a black line (b) Mediator as a repressor If Mediator interacts with a co-repressor (colored aqua) bound
to a silencer and then binds the repressive subcomplex (MED12 ⬊MED13⬊CycC/CDK8), shown in pink here, it fails
to recruit RNA polymerase II and the GTFs to the promoter, and expression from the promoter is repressed.
(Adapted from Figures 1 and 2 in Björklund, S., and Gustafson, C M., 2005 The yeast Mediator complex and its regulation Trends
Trang 10complex of RNA polymerase II, GTFs, and associated proteins that assemble at the promoter just prior to transcription initiation Mediator apparently acts as the gate-way by which RNA polymerase II gains access to the promoter As a protein kinase, Mediator also acts in phosphorylation of the unphosphorylated RNA polymerase CTD, thereby prompting its transition into the elongation phase of transcription
Mediator as a Repressor of Transcription
Mediator apparently acts in repression of transcription as well as activation How can Mediator serve two opposing regulatory functions? Perhaps the explanation lies
in the ability of a Mediator subcomplex (the MED12 and MED13 proteins, plus CycC/CDK8) to interact with co-repressor bound to a silencer (see Figure 29.29) This Mediator subcomplex is a repressive module that stabilizes Mediator in a con-formation that cannot recruit RNA polymerase II to the promoter (Figure 29.29b)
Chromatin-Remodeling Complexes and Histone-Modifying Enzymes Alleviate the Repression Due to Nucleosomes
The central structural unit of nucleosomes, the histone “core octamer” (see Figure
11.26), is constructed from the eight histone-fold protein domains of the eight various
histone monomers comprising the octamer Successive histone octamers are linked via histone H1, which is not part of the octamer Each histone monomer in the core octamer has an unstructured N-terminal tail that extends outside the core oc-tamer Interactions between histone tails contributed by core histones in adjacent nucleosomes are an important influence in establishing the higher orders of
chromatin organization Activation of eukaryotic transcription is dependent on two
sets of circumstances: (1) relief from the repression imposed by chromatin struc-ture and (2) interaction of RNA polymerase II with the promoter and transcription regulatory proteins Relief from repression requires factors that can reorganize the chromatin and then alter the nucleosomes so that promoters become accessible
to the transcriptional machinery Two sets of factors are important:
chromatin-remodeling complexes that mediate ATP-dependent conformational
(noncova-lent) changes in nucleosome structure and histone-modifying enzymes that
intro-duce covalent modifications into the N-terminal tails of the histone core octamer Chromatin remodeling and histone modification are closely linked processes
Chromatin-Remodeling Complexes Are Nucleic Acid–Stimulated Multisubunit ATPases
Chromatin-remodeling complexes are huge (1 megadalton) assemblies containing ATP-dependent enzymes that loosen the DNA⬊protein interactions in nucleosomes
by sliding, ejecting, inserting, or otherwise restructuring core octamers In the process, about 50 bp of DNA are “peeled” away from the edge of the nucleosome, creating a “bulge” that allows RNA polymerase II, GTFs, and other transcription fac-tors to access the DNA Chromatin-remodeling complexes contain proteins of the
signifies the Asp-Glu-Ala-Asp tetrapeptide signature sequence of this protein family;
in some of these proteins, a histidine residue (H) replaces the second D in the box.)
SNF2 family members include SWI2 types with bromodomains that bind acetylated
lysines, ISWI types that have separate domains for histone tail and linker DNA
bind-ing, CHD types with chromodomains that bind methylated lysines, and INO80 types
with DNA-binding domains
During the elongation phase of transcription, RNA polymerase II must pass by each nucleosome, and such passage is believed to result in the loss and regain of an
H2A/H2B dimer from the core octamer FACT (for facilitates chromatin
transcrip-tion) is a heterodimer that acts on some nucleosomes to catalyze H2A/H2B removal
and readdition; its activity is markedly increased by ubiquitination (see Chapter 31)
of H2B Lys120