Chapter 12: Mechanisms of Transcription
I. Introduction
A. Introduction to Transcription: What is Transcription?
1. In its simplest, transcription is the first step in the process of
gene expression
2. In transcription a copy of the gene in the form of an RNA
a. The RNA produced will have roughly the same sequence
as the coding strand in DNA
b. Prokaryotes produce an mRNA copy
c. Eukaryotes produce a pre-mRNA copy
3. When transcribing a gene, multiple RNA copies are going to be
produced (different than DNA replication where only one copy is
produced)
II. RNA Polymerases
A. RNA Polymerases: An Introduction To The Enzyme(s) That Catalyze
Transcription
1. Transcription is catalyzed by RNA polymerase enzymes
2. RNA polymerases are multi-subunit enzyme with a crab clawlike structure
a. Composed of multiple peptides
b. Peptides are located near the enzyme core are
important for DNA binding
c. Peptides are located near the periphery and are
involved in other interactions
3. Prokaryotes have a single RNA polymerase which transcribes
all RNA types
4. Eukaryotes have three different RNA polymerases, each
transcribes only specific RNA types
a. RNAP I (Pol I) which transcribes the 28S RNA gene
b. RNAP II (Pol II) which transcribes protein coding genes
c. RNAP III (PolIII) which transcribes tRNA, snRNA,
snoRNA genes as well as the 5S rRNA
5. The bacterial RNA polymerase most resembles RNA
polymerase II from eukaryotes structurally
B. RNA Polymerases: The Structure of The Enzyme That Catalyzes
Transcription
1. Each RNA polymerase contains several common characteristic
features of enzymes
a. Active site
b. Regulatory sites on outer binding surfaces
2. The pincers of the crab-claw structure are composed of the
largest subunits
a. Β and β’ for prokaryotes
b. RBP1 and RBP2 for eukaryotes
C. RNA Polymerases: The Structure of The Enzyme That Catalyzes
Transcription
1. The crab-claw structure allows the enzyme to incorporate the
following into the active site of the enzyme
a. DNA template (substrate)
b. Ribonucleotides
c. The RNA that is being produced (product)
2. The active site works via the two metal ion-catalytic
mechanism for nucleotide addition
a. The active site contains one Mg2+
b. The other Mg2+ is brought in with each new nucleotide
to be added
3. Mg2+ ions stabilize the nucleotide to be added in the active site
for a short period of time
a. Allows the condensation reaction to occur long enough
for condensation reaction to occur
b. A phospho-diester bond to form
D. RNA Polymerases: Introduction To Mechanism of Action
1. Functional characteristics of RNA polymerases
a. Produce many copies
b. Can be somewhat error prone (more than DNA
polymerases)
2. RNA polymerases are still fairly accurate
a. One mistake per every 10,000 nucleotides
b. DNA polymerases are even more accurate at one
mistake per every 10,000,000 bases)
3. When RNA polymerase synthesizes an RNA it does not remain
base paired to the template DNA strand
a. RNA polymerase displaces the growing RNA chain 3-5
nucleotides (5’) to the newly added ribonucleotide
b. Increases transcriptional efficiency by allowing RNA
polymerases to follow one another
III. Promoter Structure
A. The Structure Of A Promoter: The RNA Polymerase II Core Promoters
1. The eukaryotic promoter for protein coding genes lies
between the -100 bp - +35 bp
a. Core Promoter
b. Regulatory sequences
2. The Core Promoter:
a. Generally 40-60 nucleotides long and extends upstream
and downstream from the transcription start site
b. Minimal amount of promoter sequence to initiate
transcription
3. The following elements are located in Pol II promoters and are
bound by specific proteins
a. BRE (TFIIB recognition element)
b. TATA Box
c. Initiator (Inr)
d. Downstream promoter elements known as the DPE, DCE
and MTE)
B. The Structure Of A Promoter: The RNA Polymerase II Core Promoters
1. Core promoters have some, but not all of the elements
2. Core promoters can have
a. DPE with the consensus sequence GGGCGCCC or
CCACGCCC (less common)
b. TATA Box (more common) in conjunction with a DCE
(Downstream Core Element)
c. Will not have both
3. Initiator (Inr) elements are generally found in all eukaryotic
core promoters
C. The Structure Of A Promoter: Promoter Regulatory Elements
1. Regulatory elements are located upstream of the core
promoter and bind proteins that regulate transcription
a. Found between -100 bp and -35 bp
b. Promoting efficient transcription
c. Repression of transcription
2. Three different regulatory elements that promote efficient
transcription are as follows:
a. Promoter proximal elements
b. Upstream activator sequences (UAS)
c. Enhancers
3. Three different regulatory elements that act to repress
transcription are as follows:
a. Silencers
b. Boundary elements
c. Insulators
IV. Building a Transcription Pre-Initiation Complex
A. Building A Transcription Pre-Initiation Complex: Introduction
1. Proteins that bind core promoter elements are general
transcription factors
2. General Transcription factors are involved in initiating
transcription of most protein coding genes
3. General transcription factors play three significant roles in
initiating transcription
a. Help RNA polymerase II to bind the promoter
b. Melt the DNA
c. Help the RNA polymerase II to escape the promoter and
elongate the transcript
4. Transcription is mediate by forming the pre-initiation complex
on the core promoter
a. Complete set of general transcription factors plus
b. Recruited RNA polymerase II
B. Building A Transcription Pre-Initiation Complex: The Binding Of The
General Transcription Factors
1. Formation of the pre-initiation complex occurs on the TATA
box
a. Located about 30 bp upstream of the transcriptional
start site
b. Bound by the general transcription factor TFIID
2. TFIID is critical for Pre-initiation complex assembly, without it
the pre-initiation complex fails to form
3. TFIID is a multi-subunit protein
a. TBP subunit which binds the TATA box
b. TAFs (TBP Associated Factors)-some of which may
recognize other core promoter elements
4. Building A Transcription Pre-Initiation Complex: The Binding
Of The General Transcription Factors
a. TBP recognize the minor groove of the TATA element
b. Unexpected-most sequence specific binding proteins
recognize the major groove
c. Minor groove recognition is necessary for DNA
distortion
5. TBP subunit DNA binding causes the minor groove to widen
and flatten
6. TFIID provides a platform for other general transcription
factors and in the end the RNA polymerase II to bind
C. Building A Transcription Pre-Initiation Complex: The Binding Of The
General Transcription Factors
1. The rest of the general transcription factors and RNA
polymerase II are recruited in the following order
a. TFIIA
b. TFIIB
c. TFIIF and RNA polymerase (II)-stabilizes pre-initiation
complex
d. TFIIE
e. TFIIH
2. Once all of these components are bound, the promoter is
melted
a. Melting is carried out by the helicase TFIIH
b. Requires the use of ATP
D. Building a Pre-Initiation Complex: RNA Polymerase Escape From The
Promoter
1. Promoter escape must occur for RNA polymerase II to start
transcribing a pre- mRNA
2. Promoter escape by RNA polymerase II involves two steps
a. ATP hydrolysis
b. Phosphorylation of the RNA polymerase
3. The largest subunit of RNA Polymerase II enzyme has a long
carboxy terminal domain (referred to as CTD)
a. CTD contains multiple copies of a heptapeptide
sequence (NH2-Tyr-Ser-Pro-Thr-Ser-Pro-Ser)
b. Yeast has 27
c. C. elegans has 32
d. Drosophila has 45
e. Humans have 52
E. Building a Pre-Initiation Complex: RNA Polymerase Escape From The
Promoter
1. Within the heptapeptide sequence, the threonines and serines
can be phosphorylated by a number of enzymes
2. Phosphorylation results in the RNA polymerase II enzyme:
a. Becomes unbound from the most of the general
transcription factors
b. Leaves the promoter and starts the elongation phase of
transcription
V. Transcriptional Elongation
A. Transcriptional Elongation: Other Proteins Are Necessary For
Efficient Elongation
1. Elongation results in RNA Polymerase II enzyme transcribing a
pre-mRNA
a. RNA polymerase II elongate the new pre-mRNA in the 5’
 3’ direction
b. RNA polymerase II moves along the non-coding strand
in the 3’  5’ direction
2. In the elongation phase two set of proteins are recruited to the
RNA polymerase II
a. Proteins that allow for stimulation of elongation
b. Proteins required for RNA processing
3. The following proteins recruited to the RNA Polymerase II
enzyme for efficient elongation are as follows
a. TFIIS
b. hSPT5
c. pTEFb
d. TAT-SF1
e. ELL
4. pTEFb is a kinase that can stimulate elongation in three ways
a. Phosphorylates the serine in the second position of the
heptapeptide repeat which allows for elongation to occur
b. Activates another elongation factor hSPT5
c. Recruits TAT-SF1
B. Transcriptional Elongation: Other Proteins Are Necessary For
Efficient Elongation
1. RNA Polymerase II does not perform elongation at a constant
rate
a. Certain template sequences result in slowing of
elongation rate
b. Certain template sequences result in pausing
2. Both ELL and TFIIS promote efficient elongation by limiting the
pauses
a. ELL acts to suppress transient pausing
b. TFIIS acts to reduce the amount of time an RNA
polymerase is paused on the template
C. Transcriptional Elongation: Modulating Chromatin Structure Via
FACT Complex
1. Supplemental Figure: Transcriptional Elongation: Modulating
Chromatin Structure Via FACT Complex
2. Most DNA is packaged such that it is wound into chromatin
a. DNA is associated with nucleosomal components
b. DNA winding inhibits progression of the RNA
polymerase II (and associated factors) during
transcription
3. FACT complex (Facilitates Chromatin Transcription) is a
heterodimer
a. Heterodimer composed of Spt16 and SSRP1
b. Modulates chromatin structure to allow for progression
of RNA polymerase II
4. Each FACT subunit will bind to a component of the core
nucleosome
a. Spt16 binds to the H2A/H2B heterodimers
b. SSRP1 binds the H3/H4 tetramer
5. FACT modulates chromatin structure by dismantling
nucleosomes in the path of the elongating RNA Polymerase II
enzyme
a. Dismantles nucleosomes by removing one H2A/H2B
heterodimer
b. Reassembles nucleosomes by re-incorporating the
H2A/H2B heterodimer once RNA polymerase II has passed
VI. Pre-mRNA Processing
A. Pre-mRNA Processing: The RNA Polymerase II Enzyme Recruits PremRNA Processing Enzymes
1. Pre-mRNA processing occurs co-transcriptionally
2. The RNA Polymerase II CTD binds proteins involved in pre
-mRNA processing
3. The three processes involved in pre-mRNA processing are as
follows:
a. pre-mRNA Splicing
b. 5’ Cap addition
c. 3’ Poly(A) tail addition
4. pre-mRNA splicing is a more complex process than the other
two and is coupled to transcription
a. Involves a whole host of proteins
b. UsnRNAs
5. Some of the splicing machinery is also recruited to the RNA
polymerase II CTD by TAT-SF1
B. Pre-mRNA processing: 5’ Cap Addition
1. The first pre-mRNA processing step that occurs is 5’ capping
2. The 5’ capping occurs as soon as the 5’ end of the pre-mRNA
exists the Pol II active site
3. The type of cap the 5’ capping to be added to the pre-mRNA is a
5’-methyl guanosine cap
4. The 5’ methyl guanosine cap is added by series of three
enzymes
a. RNA triphosphatase
b. RNA guanylyltransferase (transfers GMP from GTP to
the diphosphate end of the RNA to form The Gppn-Cap
c. RNA guanine-7-methyl transferase (adds a methyl group
5. All three enzymes are considered the “capping enzyme” and
are recruited to the pre-mRNA as well as having their activities
stimulated by hSPT5
6. The first step in cap addition is removal of a phosphate group
from the 5’ end of the RNA by the RNA triphosphatase enzyme
7. The second step is addition of the GMP moiety to the βphosphate of the first nucleotide by the RNA guanylyl transferase
enzyme
8. The third step is the addition of a methyl group to the nitrogen
at position 7 in the guanine ring structure
C. Pre-mRNA Processing: Poly(A) Tail Addition
1. The signal for poly(A) tail addition is located at the end of a
gene
a. Carries the AATAAA sequence, which when transcribed
is AAUAAA
b. Considered the poly(A) signal sequence
2. Poly(A) tail addition is linked to the termination of
transcription and is carried out by two proteins
a. CPSF (cleavage and polyadenylation specificity factor)
b. CstF (Cleavage stimulation factor)
3. 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
4. Once the poly-adenylation signal sequence is (AAUAAA) is
transcribed:
a. Triggers the transfer of the CPSF and CstF proteins to the
pre-mRNA
b. CPSF/CstF cleaves the pre-mRNA at the polyadenylation
signal sequence
5. Polyadenylation is initiated at the point of cleavage
a. Mediated by poly(A) polymerase (PAP)
b. PAP adds adenosines to create the poly(A) tail using
ATP as a precursor
c. PAP can add a few adenosines to up to thousands of
adenosines
6. Cleavage and polyadenylation completion
a. Final steps temporally in pre-mRNA
b. Once complete, the mRNA is mature and can be exported
to the cytoplasm
7. 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!
VII. Regulation of Transcription
A. Regulation of Transcription: Introduction
1. The process of controlling which genes are being expressed is
considered “Regulation of Gene Expression”
a. Regulation of gene expression necessary for
development as well as normal cell function
b. Not all genes will be expressed in all cells all of the time
2. One way to regulate gene expression is to regulate
transcription
a. If an mRNA is produced, then the gene is expressed
(which can be then translated to produce protein)
b. If an mRNA is not produced, then the gene is not
expressed
B. Regulation of Transcription: The Promoter and Regulatory
Transcription Factors
1. Regulation of transcription occurs at the regulatory regions of
the promoter
a. Upstream of the core promoter (between -35 and -100
bp)
b. Regulatory sequences act to control gene expression by
binding regulatory transcription factors
2. There are two types of regulatory transcription factors
a. Activating transcription factors
b. Inhibitory transcription factors
C. Regulation of Transcription: The Promoter and Regulatory
Transcription Factors
1. Regulatory transcription factors control transcription in one of
two ways
a. Recruit or block efficient RNA polymerase II binding to
the core promoter
b. Effect the winding of the DNA
2. Descriptions of regulatory transcription factors that either
block, or efficiently recruit RNA polymerase to the promoter
a. Activating transcription factors recruit and “activate”
RNA polymerase II through mediator
b. Inhibitory transcription factors often block RNA
polymerase II binding to the promoter
3. Descriptions for those regulatory transcription factors that
effect winding of the DNA
a. Activating transcription factor will promote looser
winding of the DNA
b. Inhibitory transcription factors will cause the DNA to
wind more tightly
D. Regulation of Transcription: Promoter Mutations Congenital
Erythropoietic Porphyria
1. Mutations that affect transcription can lead to severe disease
a. Generally promoter mutations
b. Generally not mutations that affect the general
transcription factors
2. Very few diseases are caused by mutations in the regulatory
promoter region
3. Congenital erythrpoietic porphyria:
a. Known as Gunther’s disease, and is very rare
b. Autosomal recessive disease
c. Caused by a mutation in the regulatory promoter
uroporphyrinogen-I synthease (URO-I) gene
4. The URO-1 gene encodes the uroporphyrinogen-I synthease
enzyme
a. Mainly expressed in the erythrocytes, teeth, bones and
skin
b. Important enzyme in the pathway for synthesizing heme
in red blood cells
5. CEP can be debilitating and has several symptoms
a. Skin photosensitivity leading to blistering
b. Severe scarring
c. Increased hair growth
d. Facial features may be damaged due to photosensitivity
e. Increased risk of bacterial infection also due to
photosensitivity
f. Can be some anemia
6. Severe cases of CEP can have onset during childhood
7. Weaker cases of CEP can have adult onset
8. The pathology of this disease is still poorly understood
9. This disease can be caused by a G  A transition in the
promoter region (nt -76)
10. This transition disrupts the binding of the activating
transcription factor GATA1
11. With respect to the URO-I gene, then there are three different
possible genotypes (U = wild-type allele; u = mutant allele)
a. UU
b. Uu
c. Uu
12. In the case of the UU individual, the individual has two wildtype alleles and makes the normal amount of URO-1 enzyme
(100%)
13. In the case of the Uu individual, the individual has one wildtype, and one mutant allele
a. URO-1 transcription from the wild-type copy is normal
b. URO-1 transcription from the mutant allele is greatly
reduced (~0% of the wild-type)
c. This person ends up making about 50-58% of the URO-1
enzyme of the UU individual, but is phenoytpically normal
14. The CEP patient has a genotype of uu
a. This patient has two mutant alleles,
b. Each allele, transcription is reduced to 0-8% of normal
c. This patient produces about 0- 16% of the URO-1
enzyme compared to the UU individual
E. Regulation of Transcription: p53 and Cancer
1. Mutations that lead to changes in amino acid sequence of
regulatory transcription factors can lead to disease, especially
cancer
2. The p53 gene
a. Probably the most important gene with respect to the
study of cancer
b. Knock-out mice have no developmental phenotype
c. Knock-out mice develop cancer early in life
3. The p53 gene is a tumor suppressor gene that encodes a
regulatory transcription factor
4. The levels of p53 protein are not always high in the cell
a. Under normal conditions (no DNA damage), p53 protein
levels are low
b. Under conditions where there is DNA damage, p53
protein levels are increased
5. p53 activates transcription of two classes of target genes
a. Genes that act to halt the cell cycle (p21)
b. Genes that promote apoptosis (DR4, DR5, BAX)
6. p53 in response to DNA damage, activates transcription of p21
a. The role of p21 is to complex with cdk2
b. The p21/cdk2 heterodimer acts to stop cell cycle
progression
7. Under severe DNA damage
a. p53 activates Mdm2,
b. Leads to apoptosis
8. In cancers, mutations in p53 actually result from DNA damage
9. Mutations in the p53 gene allow for changes in amino acid
sequence of the p53 protein
a. Result in its inability to bind regulatory promoter
sequences
b. Activation of transcription of target genes will not occur
10. Those individuals that are born with just one mutant copy of
the p53 gene have Li-Fraumeni Syndrome
11. Li-Fraumeni syndrome:
a. Shows autosomal dominant inheritance
b. Rare-only 400 families in the US show this disorder
12. Cancers commonly found in Li-Fraumeni patients
a. Osteosarcomas
b. Brain Tumors
c. Schwannoma
d. Leomyosarcoma
–Breast Cancers
–adrenocarcinomas