Download RNA polymerase I

Bacterial gene
expression
Operons
A major difference between prokaryotes and eukaryotes is the
way in which their genes are organized.
Bacterial genes are organized into operons, or clusters of
coregulated genes. In addition to being physically close in the
genome, these genes are regulated such that they are all turned on
or off together. Grouping related genes under a common control
mechanism allows bacteria to rapidly adapt to changes in the
environment, switching from metabolizing one substrate to another
quickly and energetically efficiently.
For example
When glucose is abundant, bacteria use it exclusively as their food
source, even when other sugars are present.
However, when glucose supplies are depleted, bacteria have the
ability to rapidly take up and metabolize alternative sugars, such as
lactose. This process is called “induction”.
Operon regulation
All of the operon's genes are downstream of
a single promoter. This promoter serves as a
recognition site for the transcriptional
machinery of the RNA polymerase complex.
The operator is a special DNA sequence
located between the promoter sequence and
the structural genes that enables repression
of the entire operon, following binding by the
inhibitor protein.
All genes in an operon actually
become part of a single
messenger RNA molecule (premRNA or polycistronic mRNA),
which is subsequently
translated into individual
protein gene products.
Lactose (lac) operon
The best-studied examples of operons are from the bacterium
Escherichia coli (E. coli), and they involve the enzymes of lactose
metabolism and tryptophan biosynthesis.
The lactose (lac) operon shares many features with other
operons.
Francois Jacob and Jacques Monod won the Nobel prize for
their work describing the lac operon's structure and control
mechanisms.
By examining mutant strains of E. coli that exhibited defects in
lactose metabolism, Jacob and Monod were able to learn how the
lac operon is regulated to metabolize lactose (Jacob & Monod,
1962).
Lactose (lac) operon
The lac operon consists of three structural genes, lacZ, lacY,and
lacA.
lacZ encodes -galactosidase, an enzyme which cleaves
lactose into galactose and glucose both of which are
used by the cell as energy source.
lacY encodes a lactose permease that is part of the
transport system to bring lactose into the cell.
lacA encodes a transacetylase that rids the cell of toxic
thiogalactosides that get taken up by the permease
Lactose (lac) operon regulation
Upstream of the lac operon is the regulatory gene (I) that codes for the 38 kDa
Lac repressor. The Lac repressor is constitutively transcribed under control of its
own promoter (PI).
In the absence of lactose, the Lac repressor binds as a tetramer to the operator
DNA sequence (O).
Because the lac operator sequence overlaps with the promoter region, the Lac
repressor blocks RNA polymerase from binding to the promoter. As a consequence,
transcription of the lac operon structural genes is repressed.
Lactose (lac) operon regulation
In the presence of lactose and the absence of glucose, the lac operon is
induced. The real inducer is an alternative form of lactose called
allolactose.
Because repression of the lac operon is not
complete, there is always a very low level of the lac
operon products present (< 5 molecules per cell of
β-galactosidase), so
some lactose can be taken up into the bacterium
and metabolized.
When β-galactosidase cleaves lactose to
galactose plus glucose it rearranges a small
fraction of the lactose to allolactose.
Even a small amount of the inducer is enough to
start activating the lac operon.
Lactose (lac) operon regulation
Upon binding allolactose, the Lac repressor undergoes a conformational (allosteric) change
that reduce its operator-binding affinity to nonspecific levels, thereby relieving lac
repression.
The catabolic activator protein (CAP or CRP, for cyclic AMP receptor protein), binds to the
DNA sequence within the lac operon called the CAP site. Recruitment of RNA polymerase
requires the formation of a complex of CAP, polymerase, and DNA.
RNA polymerase begins transcription from the promoter and transcribes a common mRNA for
the three structural genes, from 5′ to 3′.
Basal transcription of the lac operon
The lac operon is transcribed if and only if lactose is present in the medium.
When provided with a mixture of sugars, including glucose, the bacteria use
glucose first. So long as glucose is present, operons such as lactose are not
transcribed efficiently. Only after exhausting the supply of glucose does the
bacterium fully turn on expression of the lac operon.
Glucose exerts its effect, in part, by decreasing synthesis of cAMP which is
required for the activator CAP to bind DNA.
Without the cooperative binding of CAP, RNA polymerase
transcribes the lac genes at a low level, called the basal level. This basal level of
transcription is determined by the frequency with which RNA polymerase
spontaneously binds the promoter and initiates transcription. The basal level is
some 20–40-fold lower than activated levels of transcription.
Lactose (lac) operon regulation
Regulation of the lac operon by Rho
The E. coli lac operon contains latent Rho-dependent terminators within the early part
of the operon.
Rho has been shown to terminate the synthesis of transcripts when the cells are
starved of amino acids.
The intragenic terminators do not function under conditions of normal expression.
In the absence of an amino acid, a segment of a transcript containing a Rho
utilization (rut) site becomes exposed, allowing Rho to bind and
terminate the partial transcript.
This is advantageous to the cell since it prevents the loss of energy in making a
transcript that will not be translated.
Gene regulatory networks
Bacterial regulatory networks have evolved to respond with
remarkable precision to environmental changes.
Alternative sigma factors coordinate the expression of different
sets of genes or operons.
• In general, organisms with more varied lifestyles have more 
factors.
• The number of  factors varies from 1 in Mycoplasma genitalia to
more than 63 in Streptococcus coelicolour.
• E. coli uses 7 alternative  factors to respond to some environmental
changes:
- expression of heat-shock proteins
- expression of flagellar genes
Gene regulatory networks
Bacteria communicate with each other through the production
of autoinducers.
• These molecules are produced at basal levels and accumulate
during growth.
• Once a critical concentration has been reached, autoinducers
can activate or repress a number of target genes for
collective responses.
• These responses can include light production, biofilm
formation, or virulence.
• Because the control of gene expression by autodinducers is
cell-density-dependent, this phenomenon has been called
quorum sensing.
The lac promoter and lacZ structural gene are
widely used in molecular biology research
Because its activity is easily detected by color reactions
and
its expression is inducible, β-galactosidase has become an
important enzyme in DNA biotechnology. It is often used
in screening strategies for bacterial colonies that have been
transformed with recombinant DNA during gene cloning
procedures.
The lac operon transcriptional machinery is widely used to
induce expression of heterologous proteins in E. coli. In the
lab, isopropylthiogalactoside (IPTG), a sulfur-containing
analog of lactose, is used as an inducer of the lac operon.
The advantage of IPTG over lactose is that IPTG interacts
with the Lac repressor and induces the lac operon but is not
metabolized by β-galactosidase. Thus, IPTG can continue
inducing the operon for longer periods of time in the
laboratory.
Transcription in
Eukaryotes
Introduction
Transcription in Bacteria
• RNA polymerase is the enzyme that
catalyzes RNA synthesis.
• Using DNA as a template, RNA
polymerase joins, or “ polymerizes, ”
nucleoside triphosphates (NTPs) by
phosphodiester bonds from 5' to 3'.
• In bacteria there is one type of RNA
polymerase and transcription and
translation are coupled (they occur
within a single cellular compartment).
• As soon as transcription of the mRNA
begins, ribosomes attach and initiate
protein synthesis.
• The whole
minutes.
process
occurs
within
Transcription and translation are uncoupled
in eukaryotes
Transcription takes place in the nucleus
and translation takes place in the
cytoplasm.
The whole process may take hours
Gene expression can be controlled at
many different levels, including:
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processing of the RNA transcript
transport of RNA to the cytoplasm
translation of mRNA
mRNA and protein stability
Transcription is mediated by:
• Sequence-specific DNA-binding transcription
factors.
• The general RNA polymerase II (RNA pol II)
transcriptional machinery.
• Coactivators and corepressors.
• Elongation factors.
Nuclear matrix
The nuclear matrix is defined as a branched meshwork of insoluble filamentous
proteins within the nucleus, somewhat analogous to the cell cytoskeleton.
However, in contrast to the cytoskeleton, the nuclear matrix has been proposed to
be a highly dynamic structure.
What forms the branching filaments remains
unknown and the exact function of this matrix
is still disputed .
General components of the nuclear matrix
include the heterogeneous nuclear
ribonucleoprotein (hnRNP) complex proteins
and the nuclear lamins (proteins meshwork
underlying the nuclear membrane).
What does the nuclear matrix do?
Proposed to serve as a structural organizer
within the cell nucleus.
Active genes are found associated with the
nuclear matrix only in cell types in which they
are expressed.
Chromosomal territories and transcription factories
Chromosome “painting” has shown that each chromosome occupies
its own distinct territory in the nucleus.
Chromosome with low gene density reside at the nuclear
periphery, whereas chromosomes with high gene density (human
chr 1,11,19) are located in the nuclear interior.
Transcription decondenses chromatin territories.
• The three-dimensional organization of chromatin within the cell nucleus plays a
central role in transcriptional control.
• There is increasing evidence that eukaryotic chromatin is organized as
independent loops.
• The formation of each loop is dependent on specific DNA sequence elements
that are scattered throughout the genome at 5–200 kb intervals.
• The DNA loops that form in decondensed regions are proposed to be associated
with transcription “factories.”
• Transcriptionally active genes also appear to be preferentially associated with
nuclear pore complex.
Eukaryotes have different types of RNA polymerase
Bacteria have one type of RNA polymerase that is responsible for
transcription of all genes.
Eukaryotes have multiple nuclear DNA-dependent RNA polymerases
and organelle-specific polymerases.
RNA polymerase II is located in the nucleoplasm and is responsible for
transcription of the majority of genes including those encoding
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mRNA,
small nucleolar RNAs (snoRNAs),
some small nuclear RNAs (snRNAs),
microRNAs.
Eukaryotes have different types of RNA polymerase
RNA polymerase I resides in the nucleolus and is responsible for synthesis of the
large ribosomal RNA precursor.
RNA polymerase III is also located in the nucleoplasm and is responsible for
synthesis of transfer RNA (tRNA), 5S ribosomal RNA (rRNA), and some snRNAs
We will focus here on regulation of transcription of protein-coding genes by RNA
polymerase II because the basic principles are the same from one polymerase to
another.
Protein-coding gene regulatory
elements
The big picture:
Gene regulatory elements are specific DNA sequences that are
recognized by transcription factors.
Transcription factors interpret the information present in gene
promoters and other regulatory elements and transmit the appropriate
response to the RNA pol II transcriptional machinery.
What turns on a particular gene in a particular cell is the unique
combination of regulatory elements and the transcription factors that
bind them.
Regulatory regions of unicellular eukaryotes such as yeast are usually only
composed of short sequences located adjacent to the core promoter.
Regulatory regions of multicellular eukaryotes are scattered over an average
distance of 10 kb of genomic DNA.
Two broad categories of regulatory elements.
– Promoter elements.
– Long-range regulatory elements.
Structure and function of promoter elements
The gene promoter is the collection of regulatory
elements that :
• Are required for the initiation of transcription.
• Increase the frequency of initiation only when
positioned near the transcriptional start site.
• The recognition site for RNA pol II general
transcription factors.
The gene promoter region includes
• Core promoter elements.
• Proximal promoter elements.
Core promoter elements
Approximately 60 bp DNA sequence overlapping the transcription start site (+1).
Serves as the recognition site for RNA pol II and the general transcription
factors.
The TATA box
First core promoter element identified in a eukaryotic protein-coding gene.
Sequence database analysis suggests the TATA box is present in only 32% of
potential core promoters.
Core promoter elements
Other core promoter elements differ from TATA box
• in their consensus sequence,
• in their position relative to the start of transcription and
• in which general transcription factors they bind
Core promoter elements
Core promoter elements
• A particular core promoter many contain some, all, or none of the
common elements.
• Promoter elements in some cases act together (synergistically) to
increase the efficiency of transcription initiation.
• The TATA box is the binding site for the TATA-binding protein
(TBP), which is a major subunit of the TFIID complex.
Promoter proximal elements
Promoter proximal elements increase the frequency of initiation
of transcription, but only when positioned near the transcriptional
start site.
YEAST
Regulation of TFIID binding to the core promoter in yeast depends
on an upstream activating sequence (UAS).
The vast majority of yeast genes contain a single UAS, which is
usually composed of two or three closely linked binding sites
for one or two different transcription factors.
Promoter proximal elements
MULTICELLULAR EUKARYOTIC
Multicellular eukaryotic genes are likely to contain several promoter proximal
elements.
Promoter proximal elements are located just 5′ of the core promoter and are
usually within 70–200 bp upstream of the start of transcription. Recognition sites
for transcription factors tend to be located in clusters.
Promoter proximal elements
• Transcription factors that bind promoter proximal
elements do not always directly activate or repress
transcription.
• Transcription factors may serve as “linking elements”
that recruit long-range regulatory elements, such as
enhancer, to the core promoter.
Long-range
regulatory elements
Structure and function of long-range regulatory
elements
Additional regulatory elements in multicellular eukaryotes
that can work over distances of 10 kb or more from the gene
promoter.
Long-range regulatory elements in multicellular eukaryotes
include
• Enhancers and silencers
• Insulators
• Locus control regions (LCRs)
• Matrix attachment regions (MARs)
Enhancers and silencers
• Usually are located 700 to 1000 bp or more away from the start of
transcription.
• The hallmark of enhancers (and silencers) is that, unlike promoter elements, they
can be downstream, upstream, or within an intron, and can function in either
orientation relative to the promoter.
• Increase (enhancer) or repress (silencer) gene promoter activity.
• Typically contain ~10 binding sites for several different transcription factors
and is 500 bp in lenght.
Insulators
Eukaryotic genomes are separated into gene-rich euchromatin
and gene-poor highly condensed heterochromatin.
Because heterochromatin has a tendency to spread into
neighbouring DNA, natural barriers to spreading are critical
when active genes are nearby.
Insulators
An insulator is a DNA sequence element, typically 300 bp to 2 kb in length, that
has two distinct functions:
• Chromatin boundary markers: an insulator marks the border between regions
of heterochromatin and euchromatin
• Enhancer or silencer blocking activity: an insulator prevents inappropriate
cross-activation or repression of neighbouring genes by blocking the action of
enhancers and silencers.
Insulator elements are recognized by specific DNA-binding proteins.
Locus control regions (LCRs)
• LCR are DNA sequences that organize and
maintain a functional domain of active chromatin
and enhance the transcription of downstream
genes.
• Prototype LCR characterized in the mid-1980s as
a cluster of DNase I-hypersensitive sites
upstream of the -globin gene cluster.
Matrix attachment regions (MARs)
• These DNA sequences attach to the
nuclear matrix and are termed either
scaffold attachment regions (SARs) or
matrix attachment regions (MARs)
• Interaction of MARs with the nuclear
matrix is proposed to organize chromatin
into loop domains and maintain
chromosomal territories.
Matrix attachment regions (MARs)
• Active genes tend to be part of looped
domains as small as 4 kb.
• Inactive regions of chromatin are
associated with larger domains of up to
200 kb.
• Typically AT rich sequences located near
enhancers in 5′ and 3′ flanking sequences.
• Confer tissue specificity and
developmental control of gene
expression.
• “Landing platform” for transcription
factors.
The general transcriptional
machinery
Components of the general transcription machinery
• RNA polymerase II
capable of synthesizing RNA and proofreading nascent
transcript.
• General transcription factors:
TFIIB, TFIID, TFIIE, TFIIF, and TFIIH. They are responsible
for promoter recognition and unwinding of
promoter DNA
• Mediator
serves as molecular bridge between the domains of various
transcription factor and RNA Pol II
Four major steps of transcription initiation
1. Preinitiation complex assembly
2.Initiation
3.Promoter clearance and elongation
4.Reinitiation
Crystal structure for Saccharomyces cerevisiae
RNA polymerase II
The enzyme complex consists of 12 subunits (Rpb1 to 12) that are highly conserved
among eukaryotes. Crystal structures have revealed that yeast RNA pol II has two
distinct structures and can be dissociated into
10 subunit catalytic core
Positively charge “cleft”
occupied by nucleic acids.
One side of cleft is
formed by a massive,
mobile “clamp.”
The active site is formed between
the clamp, a “bridge helix” and a
“wall” includes two Mg2+ binding
sites.
Crystal structure for Saccharomyces cerevisiae
RNA polymerase II
Heterodimer of Rpb4 and Rpb7.
The Rpb4/7 complex is
essential for
• Initiation from promoter
DNA
• mRNA nuclear export
• Transcription-coupled DNA
repair.
Crystal structure for Saccharomyces cerevisiae
RNA polymerase II
An additional component is the mobile C-terminal domain (CTD) of Rpb1.
The CTD is a unique tail-like feature of the largest subunit.
Consists of up to 52 heptapeptide
repeats of the amino acid consensus
sequence Tyr-Ser-Pro-Thr-Ser-ProSer.
Undergoes dynamic
phosphorylation of serine
residues at positions 2 and 5 in
the repeats.
Transcription initiation
requires an unphosphorylated
CTD, whereas elongation
requires a phosphorylated
CTD
Crystal structure for Saccharomyces cerevisiae
RNA polymerase II
An additional component is the mobile C-terminal domain (CTD) of Rpb1.
Tyr-Ser-Pro-Thr-Ser-Pro-Ser.
Preinitiation complex assembly
Preinitiation complex assembly
The first general transcription
factor to associate with
template DNA is TFIID
TFIID is a complex composed of
the TATA-binding protein (TBP)
and 14 TBP-associated factors
(TAFs).
Typically, protein recognize DNA using α-helix domain of the protein
inserted into the major groove of DNA. TBP is an exception to this
general rule!!
TBP uses an extensive region of β-sheet to recognize the minor groove of
the TATA box, (as well as some other core promoter elements) and to
distort the local DNA structure facilitating the initial opening of double
helix.
Preinitiation complex assembly
Binding of TFIID to the
promoter provides a platform to
recruit other general
transcription factors and RNA
polymerase to the promoter.
These proteins assemble at the
promoter in the following order:
TFIIB;
TFIIF together with RNA Pol II
( in a complex Mediator);
TFIIE and TFIIH which bind
downstream of RNA polymerase.
TFIIB orients the complex on the promoter
TFIIB binds to one end of TBP and to a GC-rich DNA sequence after the TATA
motif.
The TFIIB-TBP-DNA complex shows the direction for the start of
transcription and indicates which strand acts as the template.
TFIIE, TFIIF, and TFIIH binding completes the
preinitiation complex formation
RNA polymerase II joins the
assemblage in association with
TFIIF and Mediator.
TFIIE binds and recruits
TFIIH.
Promoter melting is mediated
by the helicase activity of
TFIIH.
TFIIH has both cyclin-dependent kinase activity
and helicase activity
Transcription elongation
requires a phosphorylated
CTD.
TFIIH is the kinase that
phosphorylates the CTD
of RNA pol II.
The helicase activity of
TFIIH is ATP-dependent.
Mediator: a molecular bridge
Mediator serves as a molecular bridge between the domain of various transcription
factors and RNA pol II. Mediator is expressed ubiquitously in eukaryotes.
A 20-subunit complex of about 30 proteins.
It has a conserved region associated with the CTD of RNA pol II
and variable protein subunits that interact with transcription factors
at regions distant from the core promoter (such as enhancer or
silencer region)
Initiation
Initiation
The kinase activity of TFIIH
phosphorylates the C-terminal
domain (CTD) of RNA polymerase
II
The helicase activity of TFIIH
unwinds the DNA allowing its
transcription into RNA.
The next step is a period of abortive initiation before the polymerase
escapes the promoter region (promoter clearance) and enter the
elongation phase
Abortive initiation
• RNA polymerase II synthesizes a series of short
transcripts
• As it moves, the polymerase holds the DNA strands apart
forming a transcription bubble.
• A transcript of >10 nucleotides and bubble collapse lead to
promoter clearance.
Promoter clearance and
elongation
Promoter clearance
• Requires phosphorylation of the C-terminal
domain (CTD) of RNA pol II.
• Phosphorylation helps RNA pol II to leave
behind most of the general transcription
factors.
• TFIID remains bound at the promoter and
allows the rapid formation of a new
preinitiation complex.
Transcription elongation through the
nucleosomal barrier
Most of the factors discussed so far are required for the
initiation of transcription but not for elongation.
RNA polymerase encounters a nucleosome approximately
every 200 bp.
Other factors are needed for the polymerase to move
through the nucleosomal array, including:
• FACT (facilitates chromatin transcription)
• Elongator
• TFIIS
NUCLEOSOME
Two copies of histones H2A, H2B, H3 and H4 form the protein core
(octamer) around which nucleosomal DNA is wrapped.
Within octamer,
two H3/H4 dimers associate to form a tetramer,
while the two H2A/H2B dimers associate at each end of the tetramer
in presence of DNA.
FACT promotes nucleosome displacement
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Experiments have shown that FACT mediates
displacement of an H2A-H2B dimer, leaving a
“hexasome” on the DNA.
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FACT helps to redeposit the dimer after
passage of RNA pol II.
Elongator facilitates transcript elongation
• Human Elongator is composed of six subunits,
including an histone acetyltransferase activity (HAT)
with specificity for histone H3.
• Interacts directly with RNA pol II and facilitates
transcription.
TFIIS relieves transcriptional arrest
The elongation factor, TFIIS, stimulates the overall rate of elongation by
limiting the length of time that polymerase pauses when it encounters
sequence that would otherwise tend to slow the enzyme’s progress.
It is a feature of polymerase that it does not transcribe through all
sequences at a constant rate.
Rather, it pauses periodically, sometimes for rather long periods, before
resuming transcription .
In the presence of TFIIS, the length of time that polymerase pauses at
any given site is reduced.
Proofreading
Proofreading and backtracking
•
RNA polymerase has a “tunable active site” that
switches between RNA synthesis and cleavage.
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RNA polymerization and cleavage both require metal
ion “A” (e.g. Mg2+ ) in the active site.
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The differential positioning of metal ion “B”
switches activity from polymerization to cleavage.
Backtracking
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When transcribing, if RNA pol II encounters
an arrest site, the polymerase pauses.
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The polymerase then backtracks, and with
the help of TFIIS cleaves the unpaired 3′
end of the transcript.
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Transcription then continues on past the
arrest site.
Role of TFIIS in RNA cleavage
• TFIIS is proposed to insert an acidic hairpin loop into the active center of RNA
pol II to position metal B and a
nucleophilic water molecule for RNA
cleavage.
The role of specific transcription
factors in gene regulation
Transcription factors mediate gene-specific
transcriptional activation or repression
• Transcription factors that serve as
repressors block the general transcription
machinery.
• Transcription factors that serve as
activators increase the rate of transcription
by several mechanism.
Transcription factors are modular proteins
Composed of separable, functional domains. The three major domains are
• DNA-binding domain
• transactivation domain
• dimerization domain
In addition,
transcription factors typically have
a nuclear localization sequence
(NLS), and some also have a nuclear
export sequence (NES).
DNA-binding domain motifs
The most common recognition pattern is an interaction between an helical domain of the protein and about 5 bp within the major groove
of the DNA double helix.
High affinity binding is dependent on overall 3-D shape and formation
of specific hydrogen bonds.
Loss of just a few hydrogen bonds or hydrophobic contacts from a
protein-DNA complex will usually result in a large loss of specificity.
Some of the most common DNA-binding domain motifs:
• Helix-turn-helix
• Zinc finger
Helix-turn-helix (HTH)
The first DNA-binding domain to be well characterized.
The classic HTH is composed of three core -helices.
The third helix, or “recognition helix,” typically forms the principal DNA–protein
interface by inserting itself into the major groove of
the DNA.
Zinc finger (Zif)
One of the most prevalent DNA-binding motifs.
A “finger” is formed by interspersed cysteines and/or histidines that covalently
bind a central zinc (Zn2+) ion.
The finger inserts its -helical portion into the major groove of the DNA.
The number of fingers is variable between different transcription factors.
Nuclear receptors have two fingers of a Cys2-Cys2 pattern.
Transactivation domain
Transactivation domains may work by recruiting or accelerating the
assembly of the general transcription factors on the gene promoter, but
their mode of action remains unclear.
They are often characterized by motifs
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Rich in acidic amino acids
Glutamine-rich regions
Proline-rich regions
Hydrophobic -sheets.
Dimerization domain
The majority of transcription factors bind DNA as homodimers or heterodimers. These
domains play an indirect structural role in DNA binding by facilitating dimerization of two
similar transcription factors.
Two dimerization domains that are relatively well characterized structurally are the helixloop-helix and leucine zipper motifs
Transcriptional coactivators and
corepressors
Increase or decrease transcriptional activity
without binding DNA directly.
• They bind directly to transcription factors
• They serve as scaffolds for recruitment of proteins with
enzymatic activity or
• They can have enzymatic activity themselves for altering
chromatin activity.
Two main classes of coactivators
Chromatin modification complexes.
• Multiprotein complexes that modify histones posttranslationally, in ways that allow greater access of other
proteins to DNA.
Chromatin remodeling complexes.
• Use the energy from ATP hydrolysis to change the contacts
between histones and DNA.
• Allow transcription factors to bind to DNA regulatory
elements.
Corepressors have the opposite effect on chromatin structure, making it
inaccessible to the binding of transcription factors or resistant to their actions.
Coactivators: Chromatin modification complexes
Post-translational modification of histone N-terminal
tails
The N-terminal tails of histones H2A, H2B, H3, and H4
are subject to a wide range of post-translational
modifications.
Function as master on/off switches that determine
whether a gene is active or inactive.
Four major types of modification
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Acetylation of lysines
Methylation of lysines and arginines
Ubiquitinylation of lysines
Phosphorylation of serines and threonines
Two less common types
• ADP-ribosylation of glutamic acid
• Sumoylation of lysines
Histone acetyltransferases
Histone acetyltransferase (HAT) directs
acetylation of histones at lysine residues.
Histone deacetylase (HDAC) catalyzes removal
of acetyl groups.
The addition of the negatively charged acetyl
group reduces the overall positive charge of the
histones.
Decreased affinity of the histone tails for the
negatively charged DNA.
Acetylation of lysines provides a specific binding
surface that can either recruit repressors or
activators of gene activity.
Histone methyltransferases
Histone methyltransferase (HMT) directs
methylation of histones on both lysine and
arginine residues.
Histone demethylase (LSD-1) removes
methyl groups.
The methyl groups increase the bulk of
histone tails but do not alter the electric
charge.
Histone methylation is linked to both
activation and repression of transcription.
Ubiquitin-conjugating enzymes
Ubiquitin is a 76 amino acid polypeptide that
acts as a signal for degradation.
Usually, the addition of polyubiquitin chains
targets a protein for degradation by the
proteasome
However, the addition of one ubiquitin
(monoubiquitinylation) can alter the function
of a protein without signaling its destruction
A ubiquitin-conjugating enzyme adds one ubiquitin to a lysine residue.
Isopeptidase removes ubiquitin.
Monoubiquitinylation of H2B is associated with activation or silencing.
Monoubiquitinylation of linker histone H1 leads to its release from DNA.
In the absence of the linker histone, chromatin becomes less condensed,
leading to gene activation.
Kinases
A specific kinase adds a phosphate
group to one or more serine or threonine
amino acids, adding a negative charge.
Phosphatase removes phosphate groups.
Phosphorylation of histone H3 or the
linker histone H1 is associated with the
activation of specific genes.
Coactivators: Chromatin remodeling complexes
Mediate at least four different changes in chromatin structure:
1 Nucleosome sliding: the position of a nucleosome changes on the DNA.
2 Remodeled nucleosomes: the DNA becomes more accessible but the
histones remain bound.
3 Nucleosome displacement: the complete dissociation of DNA and
histones.
4 Nucleosome replacement: replacement of a core histone with a
variant histone.
Three main families defined by a unique subunit
composition and the presence of a distinct ATPase
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SWI/SNF complex family
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ISWI complex family
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SWR1 complex family