Download Chapter 12: Mechanisms and Regulation of Transcription I

Chapter 12: Mechanisms and Regulation 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. Our goal is to make a copy of the gene in the form of an RNA
a. For prokaryotes, who have genes lacking introns, an
mRNA copy will be produced
b. For eukaryotes, who have genes containing introns, a
pre-mRNA copy will be produced
3. As we learned, the RNA produced will basically have the same
sequence as the coding strand
4. When transcribing a gene, multiple RNA copies are going to be
produced (different than DNA replication where only one copy is
produced)
B. Introduction to Transcription: How Many Genes Are Transcribed At A
Time?
1. Humans have approximately 20,000 genes across 3 billion
base pairs of DNA
2. Basically every cell in the human body will have 20,000 genes
3. Not all of these genes will be expressed in an individual cell at
a given time
a. Certain genes specific to epidermal cells will be
expressed in epidermal cells and not other cell types
b. Certain genes specific to neurons will be expressed only
in neurons
c. Certain genes specific to sex will only be expressed at a
given time in only a certain set of cells
4. One way to control gene expression is to control (or 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
II. RNA Polymerases
A. RNA Polymerases: An Introduction To The Enzyme(s) That Catalyze
Transcription
1. Whether the organism is a prokaryote or a eukaryote,
transcription is catalyzed by an RNA polymerase
2. For both prokaryotes and eukaryotes, the RNA polymerase is a
multi-subunit enzyme
a. The enzyme is composed of multiple peptides
b. Some of the proteins are located near the core and are
important in binding DNA or creating the active site
c. Other peptides are located near the periphery and are
involved in other interactions
3. Prokaryotes have just a single RNA polymerase in which the
core contains 5 different subunits
4. Eukaryotes have three different RNA polymerases
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. In structure, the bacterial RNA polymerase most resembles
RNA polymerase II from eukaryotes
B. RNA Polymerases: The Structure of The Enzyme That Catalyzes
Transcription
1. Although RNA polymerases are composed of multiple subunits,
the RNA polymerase contains several common characteristic
features of enzymes
a. Active site
b. Outer binding surfaces which can be sites of enzymatic
regulation
2. Due to evolutionary conservation, the structure of both the
prokaryotic and eukaryotic RNA polymerases are quite similar
3. The similarities are due to the fact that in each case, the
enzyme needs to synthesize an RNA on a DNA substrate by
incorporating nucleotides
4. The structure of the RNA polymerase resembles the shape of a
crab claw with the pincers being composed of the largest
subunits
a. Β and β’ for prokaryotes
b. RBP1 and RBP2 for eukaryotes
5. Between the two pincers lie the active site
6. When the RNA polymerase enzyme is transcribing a messenger
RNA three types of molecules are present inside the active site
a. DNA template (substrate)
b. RNA being produced (product)
c. Nucleotides for addition
7. 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
8. The goal of the metal ions is to stabilize the nucleotide to be
added in the active site long enough for a condensation reaction
to occur and a phosphodiester bond to form between the 5’ PO4 of
one the incoming ribonucleotide the 3’ OH at the end of the
growing chain
C. RNA Polymerases: Introduction To Mechanism of Action
1. RNA polymerases do not just produce one RNA copy of a gene,
but can produce many in a short period of time
2. Given that they produce many copies, the RNA polymerases
can be somewhat error prone (as compared to DNA replication in
which only one copy is produced)
3. RNA polymerases are nonetheless is still fairly accurate at one
mistake per every 10,000 nucleotides (as compared to DNA
polymerases which are even more accurate at one mistake per
every 10,000,000 bases)
4. When RNA polymerase synthesizes an RNA it does not remain
base paired to the template DNA strand
a. The RNA polymerase displaces the growing RNA chain
only a few nucleotides behind (5’) to where each
ribonucleotide is added
b. Release follows closely behind the site of
polymerization
c. Allows multiple RNA polymerases to follow one another
to synthesize many transcripts in a short amount of time
III. The Structure of A Gene
A. The Structure of The Gene: A Review
1. The job of RNA polymerase is to transcribe a gene
2. To understand just how RNA polymerase transcribes a gene,
we must again look at the structure of a gene
3. A gene has two basic units:
a. Promoter
b. Transcriptional Unit
4. In general the promoter lies can bind the following:
a. RNA polymerase
b. General Transcription Factors
c. Regulatory transcription factors
5. The transcriptional unit gets transcribed into an RNA and
starts its first base pair is noted by +1
B. The Structure Of A Gene: The RNA Polymerase II Core Promoters
1. As we saw, in order for a gene to be transcribed, it must have a
promoter
2. In eukaryotes, the minimal amount of promoter sequence
necessary for efficient and accurate transcriptional initiation is
the core promoter
3. The core promoter is generally 40-60 nucleotides long,
extending either upstream or downstream from the
transcription start site
4. The following elements are located in Pol II promoters, with
each responsible for binding a protein or set of proteins
a. BRE (TFIIB recognition element)
b. TATA Box
c. Initiator
d. Downstream promoter elements known as the DPE, DCE
and MTE)
5. Typically, functional core promoters have some, but not all of
the elements
6. For instance, core promoters will either have a DPE, which has
the consensus sequence GGGCGCCC or CCACGCCC or TATA Box,
but not both
7. Often, a core promoter with a TATA Box will also contain a DCE
(Downstream Core Element)
8. The initiator (Inr) is the most common element, and is found in
combination with both TATA boxes and DPEs
9. For all elements, the consensus sequences are shown in figure
12-14
C. The Structure Of A Gene: Promoter Regulatory Elements
1. Beyond the core promoter sequences are regulatory sequence
elements
2. These elements are located upstream of the core promoter and
are necessary for two aspects of transcriptional regulation
a. Promoting efficient transcription
b. Repression of transcription
3. Each type of element can be grouped into categories depending
on their location and function
4. Three different regulatory elements that promote efficient
transcription are as follows:
a. Promoter proximal elements
b. Upstream activator sequences (UAS)
c. Enhancers
5. Three different regulatory elements that act to repress
transcription are as follows:
a. Silencers
b. Boundary elements
c. Insulators
6. All of these elements function to bind proteins that influence
transcription in the appropriate manner
IV. Building A Transcription Pre-Initiation Complex
A. Building A Transcription Pre-Initiation Complex: Introduction
1. The role of the different elements in the core promoter is to
bind proteins involved in the initiation of transcription
2. The class of proteins that are going to bind the core promoter
are the general transcription factors
3. The general transcription factors are considered “general”
because they are involved in initiating transcription of most
genes
4. This is compared to regulatory transcription factors which
only influence the transcription of small number of genes
5. The general transcription factors play two 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
embark on the elongation phase
6. The complete set of general transcription factors plus the
recruited RNA polymerase is known as the pre-initiation
complex
B. Building A Transcription Pre-Initiation Complex: The Binding Of The
General Transcription Factors
1. The TATA box is located about 30 bp upstream of the
transcriptional start site and is where formation of the preinitiation complex begins
2. The TATA box is recognized by the general transcription factor
TFIID
a. TFIID is actually another multi-subunit protein
b. TBP subunit of TFIID binds the TATA box
c. The other proteins in the TFIID are considered TAFs
(TBP Associated Factors)
d. Some of the TAFs may recognize other core promoter
elements (but none as strongly as the TBP)
3. Please note that TFIID is critical for Pre-initiation complex
assembly, without it the pre-initiation complex fails to form
4. The role of TBP is to recognize the minor groove of the TATA
element and distort the DNA
a. This is unexpected because most sequence specific
binding proteins recognize the major groove
b. Minor groove recognition is necessary for DNA
distortion
5. When the TBP binds, it causes the minor groove to become
widened to an almost flat conformation
6. This allows TFIID to provide a platform for other general
transcription factors and in the end the RNA polymerase II to
bind
7. Once TFIID binds, the following general transcription factors
bind in the following order
a. TFIIA
b. TFIIB
c. TFIIF (along with the RNA polymerase II)
d. TFIIE
e. TFIIH
8. Once all of these components are bond, the promoter is able to
be melted
a. Melting is carried out by TFIIH
b. Requires the use of ATP
C. Building a Pre-Initiation Complex: RNA Polymerase Escape From The
Promoter
1.In order for RNA polymerase to start transcribing an mRNA
(begin the elongation phase) for a gene, it must be able to leave
the promoter region
2. In eukaryotes promoter escape by RNA polymerase involves
two steps
a. ATP hydrolysis
b. Phosphorylation of the RNA polymerase
3. As you remember, the RNA polymerase enzyme is a multisubunit enzyme
4. The largest subunit of the Pol II enzyme has a carboxy terminal
domain (referred to as CTD)
5. The CTD contains a heptapeptide sequence of NH2-Tyr-SerPro-Thr-Ser-Pro-Ser
6. Most Pol II enzymes contain multiple copies of the
heptapeptide repeat
a. Yeast has 27
b. C. elegans has 32
c. Drosophila has 45
d. Humans have 52
7. Within the heptapeptide sequence, the threonines and serines
can be phosphorylated by a number of enzymes (including one
that is a subunit of TFIIH)
8. The goal of the phosphorylation is to allow the Pol II enzyme to
become unbound from the most of the general transcription
factors
9. Once the Pol II enzyme becomes unbound, it will leave most of
the general transcription factors at the promoter and start the
elongation phase of transcription
V. Transcriptional Elongation
A. Transcriptional Elongation: Introduction
1. During the elongation phase, the Pol II enzyme will transcribe
a pre-mRNA with complementary sequence to the template
(which is the non-coding strand)
2. The Pol II enzyme will elongate the new pre-mRNA in the 5’ 
3’ direction
3. Therefore, during the elongation phase of transcription, RNA
polymerase II will move along the non-coding strand in the 3’ 
5’ direction
B. Transcriptional Elongation: Other Proteins Are Necessary For
Efficient Elongation
1. Once the Pol II enzyme leaves the promoter, another set of
proteins are recruited
a. Proteins that allow for stimulation of elongation
b. Proteins required for RNA processing
2. The following proteins recruited to the Pol II enzyme for
efficient elongation are as follows
a. TFIIS
b. hSPT5
c. pTEFb
d. TAT-SF1
e. ELL
3. Of note, 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
4. As the Pol II enzyme continues the elongation phase of
transcription, it does not perform the process at a constant rate
5. Oftentimes, during the process of transcriptional elongation,
the Pol II enzyme will encounter template sequences that will
cause it to either slow, or pause on the template
6. Both ELL and TFIIS promote efficient elongation by limiting the
pauses
7. ELL binds the elongating RNA polymerase II and acts to
suppress transient pausing
8. 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. Our discussion of transcriptional elongation has assumed that
the process is occurring on naked DNA
2. However, as we learned previously, most DNA is packaged
such that it is wound into chromatin
3. The winding of the DNA will inhibit progression of the Pol II
enzyme and its associated factors (proteins)
4. FACT (Facilitates Chromatin Transcription) is a heterodimer
that functions to modulates chromatin structure to allow for
progression of Pol II and subsequent transcription
5. FACT is a heterodimer composed of proteins
a. Spt16
b. SSRP1
6. Each protein that composes the FACT heterodimer will bind to
a component of the core nucleosome
a. Spt16 binds to the H2A/H2B heterodimers
b. SSRP1 binds the H3/H4 tetramer
7. FACT modulates chromatin structure by dismantling
nucleosomes in the path of the elongating Pol II enzyme
8. FACT dismantles nucleosomes by removing just 1 H2A/H2B
heterodimer
9. FACT also maintains genomic DNA structural integrity by reincorporating the H2A/H2B heterodimer once the Pol II enzyme
has passed
VI. Pre-mRNA Processing
A. Pre-mRNA Processing: The RNA Polymerase II Enzyme Recruits PremRNA Processing Enzymes
1. Besides binding proteins involved in promoting efficient
elongation, the Pol II CTD will also bind proteins involved in premRNA processing
2. Therefore, at least the start of pre-mRNA processing occurs cotranscriptionally
3. The three processes involved in pre-mRNA processing are as
follows:
a. Splicing
b. 5’ Cap addition
c. 3’ Poly(A) tail addition
4. Proteins involved in both 5’ cap addition and 3’ poly(A) tail
addition interact with the Pol II CTD
5. RNA splicing is a more complex process than the other two and
involves a whole host of proteins and UsnRNAs, and is coupled to
transcription in other ways
6. However, splicing machinery is also recruited to the RNA
polymerase II enzyme by TAT-SF1 and occurs cotranscriptionally
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 5’ Cap is added to the 5’ end of the RNA in three steps
7. The first step in cap addition is removal of a phosphate group
from the 5’ end of the RNA by the RNA triphosphatase enzyme
8. The second step is addition of the GMP moiety to the βphosphate of the first nucleotide by the RNA guanylyl transferase
enzyme
9. 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. As we noted earlier, 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 Pol II CTD as it approaches the end of a gene
4. Once the AAUAAA is transcribed, this triggers the transfer of
the CPSF and CstF proteins to the pre-mRNA
5. Once the CPSF and CstF proteins are transferred to the RNA,
they act to cleave the RNA at the polyadenylation signal sequence
6. The polyadenylation step is mediated by another enzyme
called poly(A) polymerase (PAP)
7. To add adenosines to create the poly(A) tail, PAP uses ATP as a
precursor
8. Depending on the RNA, the poly(A) polymerase will add
anywhere from just a few adenosine residues up to thousands of
adenosine residues
9. The cleavage and polyadenylation steps are the final steps
temporally in the processing of an mRNA, and once they are
complete, the mRNA is mature and can be exported to the
cytoplasm
10. 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. As we talked about earlier, not all genes will be expressed in
all cells all of the time
2. In every cell, some genes are expressed and some are not
3. The process of controlling which genes are being expressed is
considered “Regulation of Gene Expression”
4. Regulation of Gene Expression is absolutely necessary for
development as well as for normal cell function
5. 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. The actual promoter for a given gene is much larger than the
core promoter, which is important for Pol II binding
2. Upstream of the core promoter (between -30 and -100 bp) lie
regulatory sequences within the promter
3. These regulatory sequences act to control gene expression by
binding proteins that can regulate transcription
4. There are two types of regulatory transcription factors
a. Activating transcription factors
b. Inhibitory transcription factors
5. The goal of these regulatory transcription factors is control
transcription
6. They can 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
7. Below are descriptions for those regulatory transcription
factors that either block, or efficiently recruit RNA polymerase to
the promoter
a. Activating transcription factors recruit RNA polymerase
to the promoter to more efficiently activate transcription
b. Inhibitory transcription factors often block RNA
polymerase binding to the promoter, thus blocking
transcription
8. Below are descriptions for those regulatory transcription
factors that effect winding of the DNA
a. This type of Inhibitory transcription factor will cause
the DNA to wind more tightly, thus blocking transcription
b. This type of activating transcription factor will promote
looser winding of the DNA facilitating transcription