TRANSCRIPTION

• When the cell needs a par�cular protein, the

nucleo�de sequence of the appropriate por�on of the
immensely long DNA molecule in a chromosome is first
copied into RNA (a process called transcrip�on). It is
these RNA copies of segments of the DNA that are
used directly as templates to direct the synthesis of
the protein (a process called transla�on).

• The flow of gene�c informa�on in cells is therefore

from DNA to RNA to protein (Figure 6–1). All cells,
from bacteria to humans, express their gene�c
informa�on in this way—a principle so fundamental
that it is termed the central dogma of molecular
biology.
• Principal among these is that RNA transcripts in
eukaryo�c cells are subject to a series of processing
steps in the nucleus, including RNA splicing, before
they are permited to exit from the nucleus and be
translated into protein.
• small bits of DNA sequence that code for protein are
interspersed with large blocks of seemingly
meaningless DNA. Some sec�ons of the genome contain many genes and others lack genes
altogether.
• Proteins that work closely with one another in the cell o�en have their genes located on
different chromosomes, and adjacent genes typically encode proteins that have litle to do
with each other in the cell.
Gene Expression: Transcrip�on and Transla�on

1. Overview of Gene Expression:

o Gene expression is the process by which cells interpret and "read out" the
instruc�ons encoded in genes to produce proteins.

o Gene expression involves two main stages: transcrip�on (copying DNA to RNA) and
transla�on (conver�ng RNA into protein).

o Cells can produce mul�ple iden�cal RNA copies from a single gene, and each RNA can
generate mul�ple iden�cal protein molecules. This enables large-scale protein
synthesis from a single gene when needed.

2. Variability in Expression:

o Efficiency Differences: Genes are transcribed and translated with varying efficiencies,
allowing cells to control the quan�ty of each protein.

o Some genes produce a high quan�ty of protein (e.g., Gene A in the example), while
others produce less (e.g., Gene B), depending on cellular needs (see Figure 6-3 for
illustra�on).

o Cells can regulate gene expression to adapt to changing condi�ons, primarily by

controlling RNA produc�on.

Transcrip�on

1. Defini�on of Transcrip�on:

o Transcrip�on is the process where a specific segment

of DNA (a gene) is copied into an RNA sequence.

o This step ini�ates gene expression by conver�ng DNA

informa�on into an RNA form.

2. Chemical Composi�on of RNA:

o RNA is similar to DNA in structure but has key

differences:

 Ribonucleo�des: RNA nucleo�des contain

ribose sugar, unlike DNA’s deoxyribose
(hence the name ribonucleic acid).

 Uracil (U) instead of Thymine (T): RNA uses

uracil in place of thymine. Uracil (U) pairs
with adenine (A) via hydrogen bonding.

 Base Pairing: RNA uses complementary

base-pairing rules similar to DNA (A pairs with U, and G pairs with C).
However, RNA can some�mes have non-standard pairings, such as G with U.
RNA Structure and Proper�es

1. Single-Stranded Nature:

o Unlike DNA, which is double-stranded, RNA is generally single-stranded in cells.

o This single-stranded nature allows RNA to fold into unique three-dimensional shapes.

2. Func�onal Implica�ons of RNA Structure:

o The folding capability of RNA enables it to form

complex structures, similar to how proteins fold.

o Some RNA molecules have specific structural roles,

and their three-dimensional shapes allow them to
perform cataly�c func�ons within the cell.

Transcrip�on of RNA from DNA

o Transcrip�on starts with the opening and unwinding

of a small region of the DNA double helix to expose
the bases of the DNA strands.

o Only one strand of the DNA serves as a template for

RNA synthesis.

2. RNA Synthesis and Base-Pairing:

o Complementary Base Pairing: The RNA nucleo�de

sequence is determined by the complementary base
pairing with the DNA template strand (A with U, G
with C).

o As in DNA replica�on, complementary nucleo�des

are added to the growing RNA chain.

o Each incoming ribonucleo�de is covalently linked to

the RNA chain, forming phosphodiester bonds
between them.

o Direc�onality: RNA is synthesized in the 5ʹ to 3ʹ

direc�on as nucleo�des are added to the 3ʹ end.
 3. Differences Between Transcrip�on and DNA Replica�on:

o RNA-DNA Associa�on: The RNA strand does not

remain bound to the DNA template a�er synthesis.
Instead, it is displaced, allowing the DNA helix to
reform behind the transcrip�on site.

o Length of Transcripts: RNA molecules are generally

shorter than DNA molecules. While a DNA
molecule can be up to 250 million nucleo�de pairs,
RNA transcripts are typically only a few thousand
nucleo�des long.

RNA Polymerase and Its Role in Transcrip�on

1. RNA Polymerase Enzyme:

o Func�on: RNA polymerase is the enzyme responsible for catalyzing RNA synthesis.

o It binds to the DNA and moves along the template strand, unwinding the helix as it
progresses.

o Catalysis: RNA polymerase facilitates the forma�on of phosphodiester bonds between

nucleo�des in the RNA chain.

2. RNA Polymerase vs. DNA Polymerase:

o Ribonucleo�des vs. Deoxyribonucleo�des: RNA polymerase links ribonucleo�des,

while DNA polymerase links deoxyribonucleo�des.

o Primer Requirement: RNA polymerase can start an RNA chain without a primer, unlike
DNA polymerase, which requires a primer.

o Error Rate and Proofreading: RNA polymerase has a higher error rate (approximately
1 in 10,000 nucleo�des) compared to DNA polymerase due to fewer proofreading
mechanisms. However, errors in RNA are less consequen�al as RNA does not store
gene�c informa�on permanently.

3. Processivity and Accuracy:

o Con�nuous Processivity: RNA polymerase is highly processive, meaning it synthesizes

the en�re RNA molecule without dissocia�ng from the DNA template.

o Proofreading Mechanism: Although not as precise as DNA polymerase, RNA

polymerase has a modest proofreading ability, where it can backtrack and correct an
incorrect ribonucleo�de by an excision reac�on.

Mechanics of RNA Synthesis

1. RNA Synthesis Process:

 o RNA polymerase moves along the DNA, crea�ng a small, transient region where the
DNA and RNA form a DNA/RNA helix (about nine nucleo�de pairs).

o Release of RNA: A�er the RNA transcript is synthesized, it is quickly displaced from
the DNA, allowing the DNA helix to reform.

2. Energy Requirement:

o Ribonucleoside Triphosphates (ATP, CTP, UTP, GTP) are the substrates for RNA
synthesis.

o The hydrolysis of the phosphate-phosphate bonds of these nucleo�des releases

energy, which drives the polymeriza�on reac�on.

3. Mul�ple Transcripts from a Single Gene:

o Rapid Synthesis: Since RNA strands are immediately released, mul�ple RNA
polymerase molecules can transcribe the same gene simultaneously, allowing
thousands of transcripts to be generated in a short �me.

Structural Differences Between DNA and RNA Polymerases

1. Evolu�onary Origins:

o Although both enzymes catalyze template-dependent nucleo�de polymeriza�on, they

differ structurally.

o Divergent Evolu�on: X-ray crystallographic studies show that DNA and RNA
polymerases are structurally dis�nct, despite both containing a cri�cal Mg²⁺ ion at
the cataly�c site.

o These enzymes likely evolved separately in early cellular evolu�on, with different
lineages leading to modern DNA polymerases and RNA polymerases.

2. RNA Polymerase Structure and Func�on:

o RNA polymerase has a specialized structure that enables it to unwind DNA, catalyze
RNA synthesis, and release the RNA strand as it moves along the DNA.

o Figure 6-8 and 6-9: Illustrates that RNA polymerase synthesizes RNA as a single-
stranded molecule complementary to one DNA strand, using the DNA template
strand’s sequence.

RNA Structure and Folding

1. Single-Stranded Nature of RNA:

o Unlike the double-stranded DNA, RNA is single-stranded, allowing it to fold into

unique shapes.

o 3D Structure: RNA molecules can fold into specific three-dimensional structures due
to conven�onal and nonconven�onal base-pair interac�ons within the molecule.
 2. Func�onal Implica�ons of Folding:

o The ability to fold allows RNA to have structural and cataly�c roles, similar to how
proteins func�on. Some RNA molecules, like those involved in catalysis, can form
highly specific structures to perform their tasks.

Given that DNA and RNA polymerases both carry out template-dependent nucleo�de
polymeriza�on, it might be expected that the two types of enzymes would be structurally related.

However, x-ray crystallographic studies reveal that, other than containing a cri�cal Mg2+ ion at the
cataly�c site, the two enzymes are quite different. Template-dependent nucleo�de-polymerizing
enzymes seem to have arisen at least twice during the early evolu�on of cells.

1. RNA Polymerase (Pale Blue Structure):

o The large pale blue area represents RNA polymerase, the enzyme responsible for
synthesizing RNA.

o RNA polymerase moves along the DNA template, unwinding the double helix as it
progresses. This structure surrounds the DNA and provides the necessary
environment for RNA synthesis.

2. Template DNA Strand (Yellow):

o The yellow strand represents the template DNA. It’s the strand that RNA polymerase
reads to synthesize a complementary RNA strand.
 o The direc�on of the DNA template is labeled with 3' and 5' ends, indica�ng its
orienta�on.

3. Newly Synthesized RNA Transcript (Blue Line):

o The light blue line on the le� is the newly synthesized RNA transcript. This strand is
complementary to the template DNA.

o The RNA transcript grows in the 5' to 3' direc�on as RNA polymerase adds
nucleo�des.

4. Short Region of DNA/RNA Helix (Yellow and Blue Overlap):

o This area represents a brief hybrid of DNA and RNA where they form a short DNA-
RNA helix.

o Approximately nine nucleo�de pairs of DNA and RNA temporarily hybridize here. This
region allows the RNA strand to base-pair with the DNA template before detaching.

5. Downstream DNA Double Helix (Yellow):

o The segment of the yellow DNA strand to the right of the polymerase is the
downstream DNA double helix, which has not yet been unwound by RNA
polymerase.

o The direc�on of transcrip�on (movement of RNA polymerase along the DNA) is also
indicated.

6. Mg²⁺ Ion at Ac�ve Site (Red Dot):

o The red dot represents a magnesium ion (Mg²⁺) at the ac�ve site of RNA polymerase.

o Mg²⁺ is essen�al for catalysis, as it helps facilitate the addi�on of ribonucleo�des to

the growing RNA chain.

7. Ribonucleoside Triphosphate Uptake Channel (Blue Channel Leading into RNA

Polymerase):

o This channel in the blue RNA polymerase structure is the ribonucleoside triphosphate
uptake channel.

o Through this channel, incoming ribonucleoside triphosphates (ATP, UTP, CTP, and GTP)
enter RNA polymerase. These are the building blocks for the RNA strand, and they
provide the energy needed for polymeriza�on through their phosphate bonds.

TYPES OF RNAs
Transcrip�on Unit:

• In eukaryotes, a transcrip�on unit usually corresponds to one gene, producing a single RNA
or protein.

• In bacteria, a transcrip�on unit can include mul�ple genes, producing mRNA for several
proteins together (operon structure).

RNA vs. Protein Abundance:

• RNA makes up a small percentage of a cell’s dry weight, whereas proteins comprise about
50%.

• rRNA is the most abundant RNA type in cells; mRNA is only 3-5% of total RNA in mammalian
cells.

Diversity and Quan�ty of mRNA:

• The mRNA popula�on includes tens of thousands of different mRNA species.

• Each mRNA species is present in low amounts, with an average of 10-15 molecules per
species per cell.

1. Ini�a�on of Transcrip�on

• Importance of Ini�a�on:

o Transcrip�on ini�a�on is the key regulatory step in gene expression. It determines

which proteins are produced and at what rate.

• Bacterial RNA Polymerase Structure:

o Core Enzyme: The bacterial RNA polymerase core enzyme is a mul�subunit complex
that synthesizes RNA by reading the DNA template.

o Sigma (σ) Factor: An addi�onal protein subunit, the sigma factor, binds to the core
enzyme, forming the RNA polymerase holoenzyme. The σ factor is essen�al for
recognizing promoter regions on the DNA, which signal the start of transcrip�on.
 • RNA Polymerase Holoenzyme Binding:

o The RNA polymerase holoenzyme ini�ally binds weakly to DNA and slides along it.
When it encounters a promoter sequence (a specific DNA sequence indica�ng the
start of transcrip�on), the σ factor binds specifically to the DNA, causing the
holoenzyme to adhere �ghtly.

2. Promoter Recogni�on and DNA Opening

• Forma�on of the Transcrip�on Bubble:

o The �ghtly bound holoenzyme at the promoter opens up the double-stranded DNA to
expose about 10 nucleo�des of unpaired DNA, forming a transcrip�on bubble.

o The σ factor binds to the unpaired bases on one strand, stabilizing the bubble.

• RNA Chain Ini�a�on:

o The exposed DNA strand in the bubble acts as a template for complementary base-
pairing with incoming ribonucleo�des.

o The polymerase joins the first two ribonucleo�des together, ini�a�ng the RNA chain.

3. Abor�ve Ini�a�on and Scrunching Mechanism

• Scrunching Mechanism:

o As the polymerase begins to synthesize the first 10 or so nucleo�des of RNA, it uses a

“scrunching” mechanism. The enzyme remains bound to the promoter while pulling
upstream DNA into its ac�ve site, expanding the transcrip�on bubble.

o This scrunching creates stress, which can lead to abor�ve ini�a�on, where short RNA
segments are o�en released as stress is relieved, and the polymerase restarts RNA
synthesis.

• Promoter Clearance:

o A�er several rounds of abor�ve ini�a�on, the stress generated by scrunching enables
the core enzyme to release its �ght hold on the promoter DNA.

o Once the core enzyme detaches from the σ factor, the polymerase is ready to proceed
with transcrip�on.

4. Elonga�on of the RNA Chain

• RNA Synthesis and Polymerase Movement:

o The RNA polymerase moves along the DNA, adding nucleo�des to the growing RNA
strand in a stepwise manner. It advances one base pair for each added nucleo�de.

o The transcrip�on bubble expands at the front of the enzyme and contracts at the rear
as it progresses.

• Elonga�on Rate:

o Bacterial RNA polymerase synthesizes RNA at a rate of approximately 50 nucleo�des

per second.
5. Termina�on of Transcrip�on

• Encountering the Terminator Sequence:

o Transcrip�on con�nues un�l the polymerase reaches a termina�on signal. This signal
is typically a sequence of A-T pairs in the DNA, followed by a region with twofold
symmetry.

• Forma�on of the RNA Hairpin:

o The symmetric sequence in the DNA, when transcribed into RNA, folds into a hairpin
structure through complementary base-pairing.

o This hairpin structure disrupts the interac�on between the RNA transcript and the
RNA polymerase ac�ve site, helping to release the newly synthesized RNA.

• RNA Release and Polymerase Reassocia�on:

o A�er termina�on, the RNA polymerase releases both the DNA template and the RNA
transcript.

o The free RNA polymerase core enzyme then binds with a new σ factor to form
another holoenzyme, ready to start a new cycle of transcrip�on.

6. Conforma�onal Changes and Enzyme Tightening

• Role of Conforma�onal Changes:

o During transcrip�on ini�a�on, RNA polymerase and DNA undergo several

conforma�onal adjustments.

o These changes help open and posi�on the DNA in the polymerase ac�ve site and
ensure �ght binding so that the enzyme does not dissociate prematurely.

• Ensuring Transcrip�on Completeness:

o If RNA polymerase dissociates before comple�ng transcrip�on, it must restart the

process from the promoter.

7. Importance of RNA Folding in Termina�on

• RNA Structure and Gene Decoding:

o The folding of RNA into specific structures, such as hairpins, plays a crucial role in
various transcrip�on stages, including termina�on. This structural folding influences
how effec�vely RNA polymerase reads and decodes gene�c informa�on.
Ini�a�on Complex Forma�on (Step
1 in Figure):

• Components Involved: RNA

polymerase core enzyme
(light blue) and σ (sigma)
factor (purple).

• Func�on of σ Factor: The σ

factor helps RNA
polymerase recognize and
bind to specific DNA
sequences, known as
promoters.

• Mechanism:

o The RNA polymerase

holoenzyme (core
enzyme + σ factor)
slides along the
DNA, weakly
associa�ng with it
un�l it encounters a promoter.

o At the promoter, the holoenzyme binds �ghtly due to specific interac�ons between
the σ factor and the DNA, forming a closed complex.

2. Opening of the DNA Double Helix (Step 2):

• Complex Forma�on: Upon binding to the promoter, the holoenzyme undergoes a

conforma�onal change.

• Result: The DNA double helix unwinds, crea�ng an open complex.

• Transcrip�on Bubble:

o A small region (about 10 nucleo�des) of the DNA strands separates, crea�ng a

transcrip�on bubble.

o The σ factor stabilizes the unpaired DNA strands by binding to one of them, preparing
the template strand for RNA synthesis.

3. Abor�ve Ini�a�on (Step 3):

• Ini�al RNA Synthesis: RNA polymerase begins synthesizing a short RNA strand.

• Scrunching Mechanism:

o RNA polymerase pulls addi�onal upstream DNA into its ac�ve site without moving
forward, which creates tension in the transcrip�on bubble.
 • Abor�ve Cycles:

o The ini�al RNA transcripts are o�en short and may be released prematurely. This
process is called abor�ve ini�a�on.

o RNA polymerase may repeat this process several �mes before it successfully
synthesizes an RNA strand long enough to con�nue.

4. Promoter Clearance and Release of σ Factor (Step 4 and Step 5):

• Breaking Free: The stress generated from scrunching aids RNA polymerase in overcoming its
interac�on with the promoter.

• Release of σ Factor:

o Once RNA polymerase synthesizes around 10 nucleo�des of RNA, it detaches from the
promoter.

o The σ factor is released, allowing RNA polymerase to transi�on from the ini�a�on to
the elonga�on phase.

5. Elonga�on Phase (Step 6):

• RNA Synthesis:

o RNA polymerase moves along the DNA template, adding nucleo�des to the growing
RNA strand in a 5' to 3' direc�on.

o The transcrip�on bubble con�nues to move forward with RNA polymerase, with DNA
strands re-pairing behind it.

• Rate of Elonga�on: RNA polymerase synthesizes RNA at a rate of approximately 50

nucleo�des per second in bacteria.

6. Termina�on Signal Recogni�on (Step 7):

• Termina�on Signal: RNA polymerase eventually encounters a specific DNA sequence, known
as a terminator.

• Termina�on Hairpin:

o This sequence o�en contains A-T rich regions that, when transcribed into RNA, form a
hairpin structure (inverted repeat sequence in RNA).

o The hairpin destabilizes the RNA-DNA hybrid within RNA polymerase, leading to
termina�on.

7. Release of RNA and Reassocia�on with σ Factor (Step 7 and 8):

• RNA Release:

o RNA polymerase releases both the newly synthesized RNA transcript and the DNA
template.

o The RNA transcript is free to undergo further processing or transla�on, depending on

cellular requirements.

• Reassocia�on with σ Factor:

 o The core enzyme of RNA polymerase dissociates and re-associates with a free σ factor.

o The holoenzyme is now ready to start another round of transcrip�on on a new gene
or promoter.

A consensus nucleo�de sequence is derived by comparing many sequences with the same basic
func�on and tallying up the most common nucleo�des found at each posi�on. It therefore serves as
a sum mary or “average” of a large number of individual nucleo�de sequences.

1. Types of RNA Polymerases in Eukaryotes

• Eukaryo�c nuclei contain three types of RNA polymerases (I, II, and III), unlike bacteria,
which have only one.

o RNA Polymerase I: Transcribes 5.8S, 18S, and 28S rRNA genes.

The 5.8S, 18S, and 28S rRNA molecules are ribosomal RNA (rRNA) components of
the eukaryo�c ribosome

o RNA Polymerase II: Transcribes all protein-coding genes, as well as snoRNA genes,
miRNA genes, siRNA genes, lncRNA genes, and most snRNA genes.

o RNA Polymerase III: Transcribes tRNA genes, 5S rRNA genes, some snRNA genes, and
other small RNA genes.

2. Structural Similari�es and Differences

• RNA Polymerase II in eukaryotes shares structural similari�es with bacterial RNA

polymerase but has dis�nct func�onal differences:

o Requirement of General Transcrip�on Factors: Unlike bacterial RNA polymerase,

which requires only the σ factor, RNA polymerase II needs mul�ple transcrip�on
factors, collec�vely called general transcrip�on factors.

o Chroma�n Structure: Eukaryo�c DNA is packaged in nucleosomes and higher-order

chroma�n structures, complica�ng transcrip�on compared to bacterial DNA, which
is not packed in nucleosomes.

3. General Transcrip�on Factors (GTFs) for RNA Polymerase II

• General Transcrip�on Factors are essen�al for correctly posi�oning RNA polymerase II at
the promoter. They also help separate the DNA strands for transcrip�on and release RNA
polymerase to ini�ate elonga�on.

o These transcrip�on factors are denoted as TFIIA, TFIIB, TFIIC, TFIID, etc. (TFII stands
for “Transcrip�on Factor for Polymerase II”).

o General transcrip�on factors func�on similarly to the σ factor in bacteria, aiding in the
ini�al stages of transcrip�on.

4. Assembly of General Transcrip�on Factors at the Promoter

• The transcrip�on process begins with the binding of TFIID to the TATA box, a specific DNA
sequence rich in T and A nucleo�des.
 o The TATA box is generally located 25 nucleo�des upstream of the transcrip�on start
site.

o The TATA-binding protein (TBP), a subunit of TFIID, recognizes the TATA box and binds
to it, distor�ng the DNA as a landmark for the promoter.

• This distor�on facilitates the subsequent steps of protein assembly.

5. Step-by-Step Process of Transcrip�on Ini�a�on by RNA Polymerase II

Step 1: Binding of TFIID to the TATA Box

• TFIID, via its TBP subunit, binds to the TATA box, causing a significant DNA distor�on.

• This distor�on acts as a landmark, enabling other transcrip�on factors to recognize the
promoter region and begin assembling.

Step 2: Assembly of TFIIB

• TFIIB binds adjacent to TFIID, interac�ng with BRE elements in the promoter, which
posi�ons RNA polymerase II at the transcrip�on start site.

Step 3: Binding of TFIIF and RNA Polymerase II

• TFIIF binds to RNA polymerase II and helps stabilize its interac�on with TFIID and TFIIB,
preparing the complex for transcrip�on ini�a�on.

Step 4: Addi�on of TFIIE and TFIIH

• TFIIE and TFIIH are recruited to the complex.

o TFIIE aids in the recruitment and regula�on of TFIIH.

o TFIIH is a complex enzyme that performs several func�ons:

 It contains helicase ac�vity that uses ATP hydrolysis to unwind the DNA at
the transcrip�on start point, exposing the template strand.

 TFIIH also phosphorylates the C-terminal domain (CTD) of RNA polymerase

II, allowing it to detach from the general transcrip�on factors and begin the
elonga�on phase.

Step 5: Phosphoryla�on of the C-Terminal Domain (CTD)

• TFIIH phosphorylates Ser5 in the CTD of RNA polymerase II. This phosphoryla�on triggers:

o The release of RNA polymerase II from the general transcrip�on factors, enabling it to
proceed to elonga�on.

o CTD phosphoryla�on also recruits RNA-processing enzymes that atach to RNA

polymerase II and process the newly synthesized RNA transcript.

6. Transi�on to Elonga�on Phase

 • A�er the transcrip�on ini�a�on complex is assembled, RNA
polymerase II undergoes conforma�onal changes, which
allow it to begin synthesizing RNA without dissocia�ng from
the DNA.

• General Transcrip�on Factors are released from the DNA

and can par�cipate in another transcrip�on round, as RNA
polymerase II proceeds along the DNA template,
synthesizing RNA.

TATA Box and TFIID Binding (Part A - Green and Brown)

• At the top of the image (part A), you can see a small
rectangular TATA box (green) in the DNA. This TATA box is a
specific DNA sequence that signals where transcrip�on
should start.

• TFIID (brown) binds to this TATA box. A part of TFIID called

the TATA-binding protein (TBP) specifically recognizes and
ataches to this sequence. The binding of TFIID causes a
slight bend in the DNA, making it accessible for other factors
to bind.

Binding of TFIIB (Part B - Light Brown)

• In part B, TFIIB (light brown) ataches to the complex a�er

TFIID has bound to the TATA box. TFIIB helps posi�on RNA
Polymerase II correctly at the promoter, se�ng up the site
for transcrip�on to start.

 RNA Polymerase II and Other Factors Assemble (Part C - Various

Colors)

• Part C shows RNA Polymerase II (blue) being posi�oned at

the DNA.

• Other general transcrip�on factors, including TFIIE (orange), TFIIF (brown), and TFIIH
(yellow), assemble around RNA Polymerase II.

• Each of these factors plays a specific role:

o TFIIF helps stabilize RNA Polymerase II’s interac�on with the DNA.

o TFIIE recruits and regulates TFIIH.

o TFIIH has an essen�al func�on in the next step.

DNA Unwinding and Phosphoryla�on (Part D - Yellow)

• In part D, TFIIH (yellow) performs two cri�cal ac�ons:

o It uses energy from ATP to unwind the DNA strands at the transcrip�on start site. This
unwinding creates a single-stranded region of DNA, which RNA Polymerase II can use
as a template.
 o TFIIH also phosphorylates the C-terminal domain (CTD) of RNA Polymerase II
(illustrated as a light blue tail extending from RNA Polymerase II). This
phosphoryla�on changes the polymerase’s structure, freeing it from the general
transcrip�on factors so it can begin transcribing the DNA into RNA.

Beginning of Transcrip�on (Part E - Blue and Yellow)

• In the last part (E), RNA Polymerase II starts synthesizing RNA.

• As indicated by the yellow phosphate groups on the CTD tail, phosphoryla�on has occurred,
allowing RNA Polymerase II to enter the elonga�on phase, where it will read the DNA
template and synthesize RNA

Transcrip�on Ini�a�on by RNA Polymerase II in Eukaryotes

Step 1: Chroma�n Structure and Complexity

• Eukaryo�c DNA is �ghtly packed into nucleosomes and organized into higher-order
chroma�n structures.

• This complex packaging makes transcrip�on ini�a�on more challenging, as it requires

addi�onal proteins to open the chroma�n and allow access to the DNA.

Step 2: Role of Transcrip�on Ac�vators

• Transcrip�on Ac�vators are proteins that bind to specific DNA sequences known as
enhancers, which are usually located far from the promoter region.

• These ac�vators help atract RNA Polymerase II to the promoter, enhancing the efficiency of
transcrip�on ini�a�on.

Step 3: Mediator Complex Assembly

• The Mediator complex acts as a bridge between the ac�vators and RNA Polymerase II,
facilita�ng communica�on between them.

• It helps coordinate the interac�ons of ac�vator proteins, RNA Polymerase II, and the general
transcrip�on factors, ensuring efficient transcrip�on ini�a�on.

Step 4: Recruitment of Chroma�n-Modifying Enzymes

• Chroma�n remodeling complexes and histone-modifying enzymes are recruited to the

promoter region.

• These enzymes modify chroma�n structure to increase DNA accessibility, allowing the
transcrip�on machinery to bind to the DNA.

Step 5: Assembly of the Pre-Ini�a�on Complex (PIC)

• Over 100 protein subunits come together at the transcrip�on start site to form the pre-
ini�a�on complex (PIC).

• The PIC includes transcrip�on ac�vators, general transcrip�on factors (e.g., TFIID, TFIIB, TFIIE,
TFIIF, TFIIH), RNA Polymerase II, Mediator, chroma�n remodelers, and histone-modifying
enzymes.

The order of assembly at the promoter site generally includes:

 • Binding of TFIID to the TATA box via its TATA-binding protein (TBP) subunit.

• TFIIB binds next, stabilizing the TFIID-DNA interac�on and posi�oning RNA Polymerase II.

• RNA Polymerase II binds along with TFIIF.

• TFIIE and TFIIH assemble last, comple�ng the PIC.

Step 6: DNA Unwinding and Phosphoryla�on of RNA Polymerase II (Key Role of TFIIH)

• TFIIH uses ATP to unwind the DNA at the transcrip�on start site, exposing the template
strand.

• TFIIH also phosphorylates the C-terminal domain (CTD) of RNA Polymerase II at Ser5
residues.

• This phosphoryla�on signals RNA Polymerase II to begin transcrip�on and helps recruit RNA
processing factors.

Step 7: Release of RNA Polymerase II from the PIC

• RNA Polymerase II must be released from the general transcrip�on factors and other
components of the PIC to start moving along the DNA.

• This release may involve the breakdown (proteolysis) of certain ac�vator proteins, freeing
RNA Polymerase II for elonga�on.

Early Transcrip�on Elonga�on and Ini�al mRNA Processing

Step 8: Early Elonga�on and Nascent RNA Synthesis

• RNA Polymerase II begins moving along the DNA template, synthesizing a nascent (newly
formed) RNA transcript.

• A�er transcribing about 25 nucleo�des, RNA Polymerase II has ini�ated the early elonga�on
phase.

Step 9: Recruitment of 5ʹ Capping Enzymes

• As the nascent RNA grows to about 25 nucleo�des, the phosphorylated CTD of RNA
Polymerase II (with Ser5 phosphoryla�on) recruits enzymes for 5ʹ capping.

• The 5ʹ cap is a 7-methylguanosine structure added to the beginning of the RNA, which will
later protect it from degrada�on and aid in transla�on ini�a�on.

Steps in the 5ʹ capping process include:

• Phosphatase removes a phosphate from the 5ʹ end of the RNA.

• Guanyl Transferase adds GMP in a 5ʹ-to-5ʹ linkage.

• Methyl Transferase adds a methyl group to the guanosine cap.

Step 10: Transi�on to Full Elonga�on (CTD Phosphoryla�on at Ser2)

• As transcrip�on progresses, RNA Polymerase II’s CTD becomes phosphorylated at Ser2
residues.

• This phosphoryla�on recruits addi�onal RNA processing factors for splicing and 3ʹ-end
processing.

1. RNA Polymerase II and C-Terminal Domain (CTD) Tail (Blue

Structure):

o RNA Polymerase II transcribes DNA into RNA. It has a

C-terminal domain (CTD) with a tail that carries
various RNA-processing proteins. The CTD contains
a sequence with 52 heptad repeats (repea�ng
groups of seven amino acids) that includes two
serine amino acids in each repeat.

o The tail is phosphorylated at specific sites to manage

the addi�on of RNA-processing proteins. These
modifica�ons ensure that RNA processing occurs
correctly and sequen�ally as the RNA emerges from
the enzyme.

2. Phosphoryla�on Sites (Yellow "P" Circles on Ser2 and Ser5):

o Ser5 phosphoryla�on (yellow circle with "5")

happens early in transcrip�on. This phosphoryla�on
allows capping proteins to bind to the CTD, which
aids in the early processing of the RNA by adding a
5' cap as soon as the 5' end of the RNA emerges.

o As transcrip�on con�nues, Ser2 phosphoryla�on

(yellow circle with "2") by a kinase enzyme occurs.
This modifica�on allows the recruitment of addi�onal splicing and 3'-end processing
proteins needed for the further processing of RNA.

3. Capping Process (Upper Part of Image):

o When Ser5 is phosphorylated, capping proteins (highlighted at the top of the image)
bind to the CTD. These proteins add a 5' cap to the emerging RNA (shown at the top
right with the RNA exi�ng from RNA Polymerase II). The capping process is important
for RNA stability and future processing steps.

4. Splicing Proteins and 3'-End Processing Proteins (Middle and Lower Sec�ons of Image):

o As Ser2 becomes phosphorylated, splicing proteins (shown in the middle of the

image) bind to the RNA polymerase, allowing splicing of the RNA transcript. Splicing
removes non-coding sequences (introns) from the RNA, which is necessary to create
a mature messenger RNA (mRNA).

o Further down, 3'-end processing proteins (highlighted in green in the lower sec�on of
the image) bind to the CTD. These proteins are responsible for the cleavage and
 polyadenyla�on of the RNA, crea�ng a mature 3' end, which stabilizes the RNA and
aids in its export from the nucleus.

5. Final Release and Reset of RNA Polymerase II:

o Once RNA Polymerase II has transcribed an en�re gene, it releases the RNA transcript
and soluble phosphatases remove the phosphate groups from Ser2 and Ser5 on the
CTD. This dephosphorylated state is essen�al for RNA Polymerase II to begin another
round of transcrip�on, as only the fully dephosphorylated form can ini�ate RNA
synthesis at a new promoter site.

Summary of Events

• Ini�a�on: Ser5 phosphoryla�on recruits capping proteins.

• Elonga�on: Ser2 phosphoryla�on recruits splicing proteins, while the RNA con�nues
elonga�ng.

• Termina�on: 3'-end processing proteins bind for RNA cleavage and polyadenyla�on.

• Recycling: Phosphatases remove phosphates from the CTD to reset RNA Polymerase II for
new transcrip�on.

Summary: Key Steps Before mRNA Processing Begins

1. Transcrip�on Ini�a�on Steps (1-7):

o Chroma�n remodeling, ac�vator binding, Mediator assembly, PIC assembly, DNA

unwinding, CTD phosphoryla�on at Ser5, and release of RNA Polymerase II.

2. Early Elonga�on Steps (8-10):

o RNA synthesis starts, 5ʹ cap is added, and CTD phosphoryla�on at Ser2 signals
readiness for splicing and 3ʹ-end processing.

RNA Splicing in Eukaryo�c Cells

1. Overview of Gene Structure in Eukaryotes
Eukaryo�c genes consist of coding sequences, called exons, and noncoding sequences, called
introns. Introns are interspersed within exons, interrup�ng the coding sequence of a gene. While
exons contain the informa�on for protein synthesis, introns must be removed to generate a
func�onal mRNA transcript that can be translated into a protein. This removal process is known as
RNA splicing. RNA splicing is a cri�cal step in the matura�on of pre-mRNA (precursor mRNA), which
is ini�ally synthesized as a primary transcript that includes both exons and introns.

2. Pre-mRNA to mRNA Processing Steps

Before pre-mRNA can become mature mRNA, it undergoes several modifica�ons:

• 5' Capping: The addi�on of a modified guanosine cap to the 5' end of the RNA, which helps
protect the RNA from degrada�on and assists in ribosome binding during transla�on.

• 3' Polyadenyla�on: The addi�on of a poly-A tail at the 3' end, which also stabilizes the RNA
molecule and aids in its export from the nucleus.

• Splicing: The removal of introns from pre-mRNA and joining of exons to form a con�nuous
coding sequence, producing mature mRNA that can be translated.

3. Splicing Mechanism: The Two-Step Transesterifica�on Reac�ons

RNA splicing is carried out by a complex process involving two sequen�al transesterifica�on
reac�ons:

• First Transesterifica�on Reac�on:

o A specific adenine nucleo�de within the intron, located at a region called the
branch-point site, ini�ates the first reac�on.

o The 2' hydroxyl (–OH) group of this branch-point adenine atacks the 5' splice site,
which is located at the boundary between the exon and intron.
 o This reac�on cuts the RNA at the 5' splice site and forms a unique 2'-5'
phosphodiester bond between the branch-point adenine and the 5' end of the
intron, crea�ng a lariat structure (a looped shape).

• Second Transesterifica�on Reac�on:

o The 3' hydroxyl (–OH) group of the upstream exon, which was freed in the first
reac�on, now atacks the 3' splice site at the boundary between the intron and the
downstream exon.

o This reac�on joins the two exons together and releases the intron as a lariat
structure.

o The lariat is later degraded, while the spliced exons form a con�nuous coding
sequence in the mRNA.

4. The Spliceosome Complex

The splicing process is catalyzed by a large molecular machine called the spliceosome. The
spliceosome is a dynamic and complex structure composed of five small nuclear ribonucleoproteins
(snRNPs)—named U1, U2, U4, U5, and U6—and numerous associated proteins. Each snRNP is made
of a small nuclear RNA (snRNA) molecule bound to specific proteins. Together, these components
enable the spliceosome to recognize splice sites, catalyze the transesterifica�on reac�ons, and
release the spliced mRNA.

• Assembly of the Spliceosome:

o The spliceosome assembles on the pre-mRNA in a stepwise manner. First, U1 snRNP

binds to the 5' splice site, and U2 snRNP binds to the branch-point site, recognizing
these sequences through base-pairing interac�ons.

o Subsequently, a preassembled U4/U6–U5 tri-snRNP complex joins, comple�ng the

spliceosome assembly.

o Through a series of structural rearrangements, U1 and U4 are released, ac�va�ng

the spliceosome for catalysis.

• Spliceosome Catalysis:

o Once ac�vated, the spliceosome catalyzes the two-step transesterifica�on reac�ons,

as described above.

o A�er splicing, the spliceosome disassembles, releasing the mRNA and the lariat
intron.