Chapter 12: Mechanisms and Regulation of

Transcription
Chapter 12: Mechanisms and Regulation of
Transcription
 Introduction to Transcription: What is
Transcription?
• In its simplest, transcription is the
first step in the process of gene
expression

• In transcription a copy of the gene in

the form of an RNA
– The RNA produced will have roughly the
same sequence as the coding strand in
DNA
– Prokaryotes produce an mRNA copy
– Eukaryotes produce a pre-mRNA copy

• When transcribing a gene, multiple

RNA copies are going to be produced
(different than DNA replication
where only one copy is produced)
 RNA Polymerases: An Introduction To The
Enzyme(s) That Catalyze Transcription
• Transcription is catalyzed by RNA polymerase enzymes

• RNA polymerases are multi-subunit enzyme with a crab

claw-like structure
– Composed of multiple peptides
– Peptides are located near the enzyme core are important for
DNA binding
– Peptides are located near the periphery and are involved in
other interactions

• Prokaryotes have a single RNA polymerase which

transcribes all RNA types

• Eukaryotes have three different RNA polymerases, each

transcribes only specific RNA types
– RNAP I (Pol I) which transcribes the 28S RNA gene
– RNAP II (Pol II) which transcribes protein coding genes
– RNAP III (PolIII) which transcribes tRNA, snRNA, snoRNA
genes as well as the 5S rRNA

• The bacterial RNA polymerase most resembles RNA

polymerase II from eukaryotes structurally
 RNA Polymerases: The Structure of The
Enzyme That Catalyzes Transcription
• Each RNA polymerase
contains several common
characteristic features of
enzymes
– Active site
– Regulatory sites on outer
binding surfaces

• The pincers of the crab-claw

structure are composed of
the largest subunits
– Β and β’ for prokaryotes
– RBP1 and RBP2 for eukaryotes
RNA Polymerases: The Structure of The Enzyme
That Catalyzes Transcription
• The crab-claw structure allows the enzyme to
incorporate the following into the active site of
the enzyme
– DNA template (substrate)
– Ribonucleotides
– The RNA that is being produced (product)

• The active site works via the two metal ion-

catalytic mechanism for nucleotide addition
– The active site contains one Mg2+
– The other Mg2+ is brought in with each new
nucleotide to be added

• Mg2+ ions stabilize the nucleotide to be added

in the active site for a short period of time
– Allows the condensation reaction to occur long
enough for condensation reaction to occur
– A phospho-diester bond to form
 RNA Polymerases: Introduction To
Mechanism of Action
• Functional characteristics of RNA
polymerases
– Produce many copies of pre-mRNA
– Can be somewhat error prone

• RNA polymerases are still fairly accurate

– One mistake per every 10,000 nucleotides
– DNA polymerases which are even more accurate
at one mistake per every 10,000,000 bases)

• When RNA polymerase synthesizes an RNA

it does not remain base paired to the
template DNA strand
– RNA polymerase displaces the growing RNA
chain 3-5 nucleotides (5’) to the newly added
ribonucleotide
– Increases transcriptional efficiency by allowing
RNA polymerases to follow one another
 The Structure Of A Gene: The RNA
Polymerase II Core Promoters
• The eukaryotic promoter for protein coding
genes lies between the -100 bp - +35 bp
– Core Promoter
– Regulatory sequences

• The Core Promoter:

– Generally 40-60 nucleotides long and extends
upstream and downstream from the transcription
start site
– Minimal amount of promoter sequence to initiate
transcription

• The following elements are located in Pol II

promoters and are bound by specific proteins
– BRE (TFIIB recognition element)
– TATA Box
– Initiator (Inr)
– Downstream promoter elements known as the
DPE, DCE and MTE)
 The Structure Of A Gene: The RNA
Polymerase II Core Promoters
• Core promoters are different for
different genes
– Have some core promoter elements
– Will not have all of the elements

• Core promoters can have

– DPE with the consensus sequence
GGGCGCCC or CCACGCCC (less common)
– TATA Box (more common) in conjunction
with a DCE (Downstream Core Element)
– Will not have both

• Initiator (Inr) elements are generally

found in all eukaryotic core
promoters
 The Structure Of A Gene: Promoter
Regulatory Elements
• Regulatory elements are located upstream
of the core promoter and bind proteins
that regulate transcription
– Found between -100 bp and -35 bp
– Promoting efficient transcription
– Repression of transcription

• Three different regulatory elements that

promote efficient transcription are as
follows:
– Promoter proximal elements
– Upstream activator sequences (UAS)
– Enhancers

• Three different regulatory elements that

act to repress transcription are as follows:
– Silencers
– Boundary elements
– Insulators
 Building A Transcription Pre-Initiation
Complex: Introduction
• Proteins that bind core promoter elements
are general transcription factors

• General Transcription factors are involved in

initiating transcription of most protein coding
genes

• General transcription factors play three

significant roles in initiating transcription
– Help RNA polymerase II to bind the promoter
– Melt the DNA
– Help the RNA polymerase II to escape the
promoter and elongate the transcript

• Transcription is mediate by forming the pre-

initiation complex on the core promoter
– Complete set of general transcription factors plus
– Recruited RNA polymerase II
 Building A Transcription Pre-Initiation Complex: The
Binding Of The General Transcription Factors
• Formation of the pre-initiation
complex occurs on the TATA box
– Located about 30 bp upstream of the
transcriptional start site
– Bound by the general transcription factor
TFIID

• TFIID is critical for Pre-initiation

complex assembly, without it the pre-
initiation complex fails to form

• TFIID is a multi-subunit protein

– TBP subunit which binds the TATA box
– TAFs (TBP Associated Factors)-some of
which may recognize other core
promoter elements
 Building A Transcription Pre-Initiation Complex: The
Binding Of The General Transcription Factors
• TBP recognize the minor groove of
the TATA element
– Unexpected-most sequence specific
binding proteins recognize the major
groove
– Minor groove recognition is necessary
for DNA distortion

• TBP subunit DNA binding causes the

minor groove to widen and flatten

• TFIID provides a platform for other

general transcription factors and in
the end the RNA polymerase II to
bind
 Building A Transcription Pre-Initiation Complex: The
Binding Of The General Transcription Factors
• The rest of the general
transcription factors and RNA
polymerase II are recruited in the
following order
– TFIIA
– TFIIB
– TFIIF and RNA polymerase (II)-
stabilizes pre-initiation complex
– TFIIE
– TFIIH

• Once all of these components are

bound, the promoter is melted
– Melting is carried out by the helicase
TFIIH
– Requires the use of ATP
 Building a Pre-Initiation Complex: RNA
Polymerase Escape From The Promoter
• Promoter escape must occur for RNA
polymerase II to start transcribing a pre-
mRNA

• Promoter escape by RNA polymerase II

involves two steps
– ATP hydrolysis
– Phosphorylation of the RNA polymerase

• The largest subunit of RNA Polymerase II

enzyme has a long carboxyl terminal domain
(referred to as CTD)
– CTD contains multiple copies of a heptapeptide
sequence (NH2-Tyr-Ser-Pro-Thr-Ser-Pro-Ser)
– Yeast has 27
– C. elegans has 32
– Drosophila has 45
– Humans have 52
 Building a Pre-Initiation Complex: RNA
Polymerase Escape From The Promoter
• Within the heptapeptide
sequence, the threonines and
serines can be phosphorylated
by a series of kinases

• Phosphorylation results in the

RNA polymerase II enzyme:
– Becomes unbound from the
most of the general
transcription factors
– Leaves the promoter and starts
the elongation phase of
transcription
 Transcriptional Elongation: Other Proteins Are
Necessary For Efficient Elongation
• Elongation results in RNA Polymerase II enzyme
transcribing a pre-mRNA
– RNA polymerase II elongate the new pre-mRNA in the 5’  3’
direction
– RNA polymerase II moves along the non-coding strand in the 3’ 
5’ direction

• In the elongation phase two set of proteins are recruited to

the RNA polymerase II
– Proteins that allow for stimulation of elongation
– Proteins required for RNA processing

• The following proteins recruited to the RNA Polymerase II

enzyme for efficient elongation are as follows
– TFIIS
– hSPT5
– pTEFb
– TAT-SF1
– ELL

• pTEFb is a kinase that can stimulate elongation in three

ways
– Phosphorylates the serine in the second position of the
heptapeptide repeat which allows for elongation to occur (on Pol II
CTD)
– Activates another elongation factor hSPT5
– Recruits TAT-SF1
 Transcriptional Elongation: Other Proteins Are
Necessary For Efficient Elongation
• RNA Polymerase II does not
perform elongation at a constant
rate
– Certain template sequences result in
slowing of elongation rate
– Certain template sequences result in
pausing

• Both ELL and TFIIS promote

efficient elongation by limiting the
pauses
– ELL acts to suppress transient pausing
– TFIIS acts to reduce the amount of
time an RNA polymerase is paused on
the template
Transcriptional Elongation: Modulating
Chromatin Structure Via FACT Complex
 Transcriptional Elongation: Modulating
Chromatin Structure Via FACT Complex
• Most DNA is packaged such that it is
wound into chromatin
– DNA is associated with nucleosomal
components
– DNA winding inhibits progression of
the RNA polymerase II (and associated
factors) during transcription

• FACT complex (Facilitates Chromatin

Transcription) is a heterodimer
– Heterodimer composed of Spt16 and
SSRP1
– Modulates chromatin structure to
allow for progression of RNA
polymerase II
 Transcriptional Elongation: Modulating
Chromatin Structure Via FACT Complex
• Each FACT subunit will bind to a
component of the core nucleosome
– Spt16 binds to the H2A/H2B
heterodimers
– SSRP1 binds the H3/H4 tetramer

• FACT modulates chromatin structure

by dismantling nucleosomes in the
path of the elongating RNA
Polymerase II enzyme
– Dismantles nucleosomes by removing
one H2A/H2B heterodimer
– Reassembles nucleosomes by re-
incorporating the H2A/H2B heterodimer
once RNA polymerase II has passed
 Pre-mRNA Processing: The RNA Polymerase II Enzyme
Recruits Pre-mRNA Processing Enzymes
• Pre-mRNA processing occurs co-transcriptionally

• The RNA Polymerase II CTD binds proteins involved

in pre-mRNA processing

• The three processes involved in pre-mRNA

processing are as follows:
– pre-mRNA Splicing
– 5’ Cap addition
– 3’ Poly(A) tail addition

• pre-mRNA splicing is a more complex process than

the other two and is coupled to transcription
– Involves a whole host of proteins
– U snRNAs

• Some of the splicing machinery is also recruited to

the RNA polymerase II CTD by TAT-SF1
 Pre-mRNA processing: 5’ Cap Addition
• The first pre-mRNA processing step that occurs is 5’
capping

• The 5’ capping occurs as soon as the 5’ end of the

pre-mRNA exists the Pol II active site

• The type of cap the 5’ capping to be added to the

pre-mRNA is a 5’-methyl guanosine cap

• The 5’ methyl guanosine cap is added by series of

three enzymes
– RNA triphosphatase
– RNA guanylyltransferase (transfers GMP from GTP to
the diphosphate end of the RNA to form The Gppn-Cap
– RNA guanine-7-methyl transferase (adds a methyl group

• All three enzymes are considered the “capping

enzyme” and are recruited to the pre-mRNA as
well as having their activities stimulated by hSPT5
 Pre-mRNA processing: 5’ Cap Addition
• The first step in cap addition is removal of
a phosphate group from the 5’ end of the
RNA by the RNA triphosphatase enzyme

• The second step is addition of the GMP

moiety to the β-phosphate of the first
nucleotide by the RNA guanylyl
transferase enzyme
– Β phosphate of the first nucleotide attacks
the α phosphate of the GTP
– Ppi is lost, and a GMP moiety is added

• The third step is the addition of a methyl

group to the nitrogen at position 7 in the
guanine ring structure by 7-methyl
guanine transferase
Pre-mRNA Processing: Poly(A) Tail Addition
• The signal for poly(A) tail addition is located at
the end of a gene
– Carries the AATAAA sequence, which when
transcribed is AAUAAA
– Considered the poly(A) signal sequence

• Poly(A) tail addition is linked to the termination

of transcription and is carried out by two
proteins
– CPSF (cleavage and polyadenylation specificity
factor)
– CstF (Cleavage stimulation factor)

• Both the CPSF and CstF proteins are recruited

and carried by the RNA polymerase II CTD as it
approaches the end of a gene -(AAUAAA
sequence)
Pre-mRNA Processing: Poly(A) Tail Addition
• Once the poly-adenylation signal sequence is
(AAUAAA) is transcribed:
– Triggers the transfer of the CPSF and CstF proteins to the pre-
mRNAto
– Cleaves the pre-mRNA at the polyadenylation signal sequence

• Polyadenylation is initiated at the point of cleavage

– Mediated by poly(A) polymerase (PAP)
– PAP adds adenosines to create the poly(A) tail using ATP as a
precursor
– PAP can add a few adenosines to up to thousands of
adenosines

• Cleavage and polyadenylation

– Final steps temporally in pre-mRNA
– Once complete, the mRNA is mature and can be exported to
the cytoplasm

• Note: although the mRNA is mature, this does not

trigger the RNA pol II enzyme to fall off the template
DNA, it keeps on transcribing!
 Regulation of Transcription: Introduction
• The process of controlling which genes
are being expressed is considered
“Regulation of Gene Expression”
– Regulation of gene expression necessary
for development as well as normal cell
function
– Not all genes will be expressed in all cells
all of the time

• One way to regulate gene expression is

to regulate transcription
– If an mRNA is produced, then the gene is
expressed (which can be then translated
to produce protein)
– If an mRNA is not produced, then the
gene is not expressed
 Regulation of Transcription: The Promoter and
Regulatory Transcription Factors
• Regulation of transcription
occurs at the regulatory regions
of the promoter
– Upstream of the core promoter
(between -35 and -100 bp)
– Regulatory sequences act to
control gene expression by
binding regulatory transcription
factors

• There are two types of

regulatory transcription factors
– Activating transcription factors
– Inhibitory transcription factors
 Regulation of Transcription: The Promoter and
Regulatory Transcription Factors
• Regulatory transcription factors control
transcription in one of two ways
– Recruit or block efficient RNA polymerase II binding to
the core promoter
– Effect the winding of the DNA

• Descriptions of regulatory transcription factors

that either block, or efficiently recruit RNA
polymerase to the promoter
– Activating transcription factors recruit and “activate”
RNA polymerase II through mediator
– Inhibitory transcription factors often block RNA
polymerase II binding to the promoter

• Descriptions for those regulatory transcription

factors that effect winding of the DNA
– Activating transcription factor will promote looser
winding of the DNA
– Inhibitory transcription factors will cause the DNA to
wind more tightly
 Regulation of Transcription: Promoter
Mutations Congenital Erythropoietic Porphyria
• Mutations that affect transcription can lead to
severe disease
– Generally promoter mutations
– Generally not mutations that affect the general
transcription factors

• Very few diseases are caused by mutations in the

regulatory promoter region

• Congenital erythrpoietic porphyria:

– Known as Gunther’s disease, and is very rare
– Autosomal recessive disease
– Caused by a mutation in the regulatory promoter
uroporphyrinogen-I synthetase (URO-I) gene

• The URO-1 gene encodes the uroporphyrinogen-I

synthetase enzyme
– Mainly expressed in the erythrocytes, teeth, bones and
skin
– Important enzyme in the pathway for synthesizing heme
in red blood cells
Regulation of Transcription: Promoter Mutation
and Congenital Erythropoietic Porphyria
• CEP can be debilitating and has several
symptoms
– Skin photosensitivity leading to blistering
– Severe scarring
– Increased hair growth
– Facial features may be damaged due to
photosensitivity
– Increased risk of bacterial infection also due to
photosensitivity
– Can be some anemia

• Severe cases of CEP can have onset during

childhood

• Weaker cases of CEP can have adult onset

• The pathology of this disease is still poorly

understood
Regulation of Transcription: Promoter Mutation
and Congenital Erythropoietic Porphyria
• This disease can be caused by a G 
A transition in the promoter region
(nt -76)

• This transition disrupts the binding

of the activating transcription factor
GATA1

• With respect to the URO-I gene, then

there are three different possible
genotypes (U = wild-type allele; u =
mutant allele)
– UU
– Uu
– Uu
 Regulation of Transcription: Promoter Mutations and
Congenital Erythropoietic Porphyria

• In the case of the UU individual, the individual

has two wild-type alleles and makes the
normal amount of URO-1 enzyme (100%)

• In the case of the Uu individual, the individual

has one wild-type, and one mutant allele
– URO-1 transcription from the wild-type
copy is normal
– URO-1 transcription from the mutant
allele is greatly reduced (~0% of the wild-
type)
– This person ends up making about 50-
58% of the URO-1 enzyme of the UU
individual, but is phenoytpically normal
 Regulation of Transcription: Promoter Mutations and
Congenital Erythropoietic Porphyria
• The last individual to study is the
CEP patient

• The CEP patient has a genotype of

uu

• This patient has two mutant

alleles, and thus for each allele,
transcription is reduced to 0-8% of
normal

• This patient makes about 0- 16% of

the URO-1 enzyme compared to
the UU individual
Regulation of Transcription: p53 and Cancer
• Mutations that lead to changes in amino acid
sequence of regulatory transcription factors can
lead to disease, especially cancer

• The p53 gene

– Probably the most important gene with respect to
the study of cancer
– Knock-out mice have no developmental phenotype
– Knock-out mice develop cancer early in life

• The p53 gene is a tumor suppressor gene that

encodes a regulatory transcription factor

• The levels of p53 protein are not always high in

the cell
– Under normal conditions (no DNA damage), p53
protein levels are low
– Under conditions where there is DNA damage, p53
protein levels are increased
Regulation of Transcription: p53 and Cancer
• P53 activates transcription of two classes of target genes
– Genes that act to halt the cell cycle (p21)
– Genes that promote apoptosis (DR4, DR5, BAX)

• p53 in response to DNA damage, activates transcription

of p21
– The role of p21 is to complex with cdk2
– The p21/cdk2 heterodimer acts to stop cell cycle progression

• Under severe DNA damage

– p53 activates Mdm2,
– Leads to apoptosis

• In cancers, mutations in p53 actually result from DNA

damage

• Mutations in the p53 gene allow for changes in amino

acid sequence of the p53 protein
– Result in its inability to bind regulatory promoter sequences
– Activation of transcription of target genes will not occur

• Those individuals that are born with just one mutant

copy of the p53 gene have Li-Fraumeni Syndrome
Regulation of Transcription: p53 and Cancer
• Li-Fraumeni syndrome:
– Shows autosomal dominant
inheritance
– Rare-only 400 families in the US
show this disorder

• Cancers commonly found in Li-

Fraumeni patients
– Osteosarcomas
– Brain Tumors
– Schwannoma
– Leomyosarcoma
– Breast Cancers
– adrenocarcinomas