Unit 3

DAYANANDA SAGAR COLLEGE OF ENGINEERING
UNIT- 3
TRANSCRIPTION
Transcription factors
Basal transcription complex formation
Eukaryotic transcriptional activators
Chromatin structure & regulation
Histone modifiers
Co-activators & Co-repressors
RNA polymerase- structure and function of RNA polymerases (prokaryotes &

eukaryotes)
Mechanism of transcription in prokaryotes and eukaryotes
Prokaryotic transcription
Eukaryotic transcription
Post-transcriptional processing (RNA editing, splicing, poly A tail and 5‘ capping,

siRNA, miRNAs, other ncRNAs)
Transcription inhibitors
Regulation of transcription in prokaryotes and eukaryotes
CELL & MOLECULAR BIOLOGY (19BT3DCCMB ) Notes by: Dr. BLESSY B MATHEW
Transcription factors
In molecular biology, a transcription factor (TF) (or sequence-specific DNA-binding factor) is a

protein that controls the rate of transcription of genetic information from DNA to messenger
RNA, by binding to a specific DNA sequence.
There are two mechanistic classes of transcription factors:

 General transcription factors are involved in the formation of a preinitiation complex. ...
 Upstream transcription factors are proteins that bind somewhere upstream of the initiation site
to stimulate or repress transcription.
Transcription factors are a very diverse family of proteins and generally function in multi-
subunit protein complexes. They may bind directly to special ―promoter‖ regions of DNA, which
lie upstream of the coding region in a gene, or directly to the RNA polymerase molecule. The
activity of inducible transcription factors can be regulated by several mechanisms, such as
phosphorylation or dephosphorylation, binding of activating or inhibitory factors, or de novo
synthesis. Transcription factors play critical roles in the development and function of the immune
system.
General transcription factors are proteins that help to position Pol II correctly on the promoter,
the region of a gene where transcription is initiated, pull aside the two strands of DNA and then
move Pol II into the elongation mode. Transcription factors are proteins involved in the process
of converting, or transcribing, DNA into RNA. Transcription factors include a wide number of
proteins, excluding RNA polymerase, which initiate and regulate the transcription of genes.
Transcription factors (TFs) in prokaryotes are proteins that bind to specific sequences on the
DNA near their target genes, thus modulating transcription initiation. TFs can activate or repress
transcription depending where they bind relative to the transcription start site of the target gene.
Transcription factors (TFs) in eukaryotes are the factors typically have DNA-binding domains
that bind specific sequence elements of the core promoter and help recruit RNA polymerase to
the transcriptional start site. General transcription factors for RNA polymerase II include TFIID,
TFIIA, TFIIB, TFIIF, TFIIE, and TFIIH.
In negative regulation a repressor protein binds to an operator to prevent a gene from being
expressed. In positive regulation a transcription factor is required to bind at the promoter in order
to enable RNA polymerase to initiate transcription. Positive transcription factors are proteins that
help turn specific genes "on" or "off" by binding to nearby DNA. Transcription factors that are
activators boost a gene's transcription.
On the other hand, translation factors function to coordinate and catalyze the synthesis of
polypeptides from mRNA templates by ribosomes.
Basal transcription complex formation
General transcription factors (GTFs), also known as basal transcriptional factors, are a class of
protein transcription factors that bind to specific sites (promoter) on DNA to activate
transcription of genetic information from DNA to messenger RNA. A transcription-initiation
complex comprises an RNA polymerase and various general transcription factors bound to the
promoter region. Many general transcription factors required for Pol II to initiate transcription
from most TATA-box promoters in vitro have been isolated and characterized.
Basal, or general, transcription factors are necessary for RNA polymerase to function at a site of
transcription in eukaryotes. They are considered the most basic set of proteins needed to activate
gene transcription, and they include a number of proteins, such as TFIIA. These transcription
factors are a very diverse family of proteins and generally function in multi-subunit protein
complexes. They may bind directly to special ―promoter‖ regions of DNA, which lie upstream of
the coding region in a gene, or directly to the RNA polymerase molecule.
Eukaryotic transcriptional activators
Transcription of eukaryotic structural genes is regulated by promoter-specific activator proteins

(activators), which are generally sequence-specific DNA binding proteins. In many cases, such
fusion proteins artificially direct the assembly of a PIC complex, resulting in transcription.
In bacteria, RNA polymerase attaches right to the DNA of the promoter. In humans and other
eukaryotes, there is an extra step. RNA polymerase can attach to the promoter only with the help
of proteins called basal (general) transcription factors. They are part of the cell's core
transcription toolkit, needed for the transcription of any gene. However, many transcription
factors (including some of the coolest ones!) are not the general kind. Instead, there is a large
class of transcription factors that control the expression of specific, individual genes.
A typical transcription factor binds to DNA at a certain target sequence. Once it's bound, the
transcription factor makes it either harder or easier for RNA polymerase to bind to the promoter
of the gene.
Activators
Some transcription factors activate transcription. For instance, they may help the general
transcription factors and/or RNA polymerase bind to the promoter, as shown in the diagram
below.
Fig. 1 Activator
Diagram of an activator attached to a specific DNA sequence that is its binding site. The other
end of the transcriptional activator (the one not bound to the DNA) interacts with general
transcription factors, helping the general transcription factors and polymerase assemble tat the
nearby promoter.
Repressors
Other transcription factors repress transcription. This repression can work in a variety of ways.
As one example, a repressor may get in the way of the basal transcription factors or RNA
polymerase, making it so they can't bind to the promoter or begin transcription.
Fig. 2 Repressors
Diagram of a repressor attached to a specific DNA sequence that is its binding site. When bound
to this site, the repressor blocks formation of the transcription initiation complex at the promoter
of a nearby gene.
Binding sites
The binding sites for transcription factors are often close to a gene's promoter. However, they
can also be found in other parts of the DNA, sometimes very far away from the promoter, and
still affect transcription of the gene.
Fig. 3 Binding sites
The parts of an activator protein: the DNA binding domain (which attaches to the recognition site
in the DNA) and the activation domain, which is the "business end" of the activator that actually
promotes transcription, e.g., by facilitating formation of the transcription initiation complex. The
flexibility of DNA is what allows transcription factors at distant binding sites to do their job. The
DNA loops like cooked spaghetti to bring far-off binding sites and transcription factors close to
general transcription factors or "mediator" proteins.
Chromatin structure & regulation
Chromatin is a dynamic structure that not only helps to package the entire eukaryotic genome
into the confines of the nucleus but also regulates the accessibility of DNA for transcription,
recombination, DNA repair and replication. Chromatin is located in the nucleus of our cells. The
major proteins in chromatin are histones, which help package the DNA in a compact form that
fits in the cell nucleus. The primary function of chromatin is to compress the DNA into a
compact unit that will be less voluminous and can fit within the nucleus.
Chromatin consists of complexes of small proteins known as histones and DNA. Chromatin is
not an inert structure, but rather an instructive DNA scaffold that can respond to external cues to
regulate the many uses of DNA. A principle component of chromatin that plays a key role in this
regulation is the modification of histones. Chromatin structure plays a key role in regulating gene
expression by allowing DNA accessibility to transcriptional machinery and transcription factors.
To form chromatin, DNA is tightly condensed by being wrapped around nuclear proteins called
histones. Epigenetic modifications to histone proteins such as methylation/demethylation and
acetylation/deacetylation can alter the structure of chromatin resulting in transcriptional
activation or repression.
Structure of chromatin:
Chromatin structure plays an important role in controlling gene expression and replication. The
packaging of DNA into nucleosomes forms a ‗closed‘ structure that is not very accessible to
enzymes that perform replication, transcription, and DNA repair. This structure is generally
transcriptionally repressive, allowing only a basal level of gene expression. In a disrupted, ‗open‘
nucleosome structure, the DNA is more accessible to replication and transcription factors.
In transcription, some activators and repressors interact with RNA polymerases to change the
chromatin structure and modulate gene activity. Activators can help to disrupt nucleosome
structure and thereby stimulate the assembly of RNA polymerase and transcription factors at the
promoter. For replication, a similar modulation of chromatin structure must occur to allow the
replication machinery to be positioned at the origins of replication.
Fig.4 Epigenetic regulation of chromatin structure
The structure of chromatin can also have long-range effects on gene expression. In a
phenomenon termed ‗position effect variegation,‘ genes located near silent heterochromatic
regions can also be made transcriptionally inactive. Genes as far as 1000 kb away can be
silenced. Because the exact areas that are repressed vary from cell to cell, this is an epigenetic
phenomenon that produces variegation in phenotype. It is generally thought that the highly
condensed nature of heterochromatin prevents access by transcription factors, but how this can
affect neighboring, nonheterochromatic regions is not fully understood. While it is accepted that
proteins found in the heterochromatin can ‗spread‘ to adjoining regions and impact a similar
repressive effect, another possibility is that the heterochromatin may be grouped into
compartments of the nucleus that are inaccessible to transcription factors.
Chromatin structure can also affect DNA replication on a global level. For example,
heterochromatin and other silent areas of the genome replicate late in S-phase, but the reason that
these late-replicating regions are silent is unknown. One possibility is a specific repressive
chromatin structure that can be overcome to allow origin firing late in S-phase.
Fig. 5 DNA packaging: Nucleosomes and chromatin
Chromatin remodeling
Chromatin remodeling is the rearrangement of chromatin from a condensed state to a

transcriptionally accessible state, allowing transcription factors or other DNA binding proteins to
access DNA and control gene expression. Chromatin remodeling is an important mechanism of
regulating eukaryotic gene expression, which makes tightly condensed DNA accessible to
various regulatory factors, such as transcription factors and components of DNA replication.
Fig.6 Gene switching on and off modes
Such remodeling is principally carried out by:
1) covalent histone modifications by specific enzymes, e.g., histone acetyltransferases (HATs),

deacetylases, methyltransferases, and kinases
2) ATP-dependent chromatin remodeling complexes which either move, eject or restructure

nucleosomes. Besides actively regulating gene expression, dynamic remodeling of chromatin
imparts an epigenetic regulatory role in several key biological processes, egg cells DNA
replication and repair; apoptosis; chromosome segregation as well as development and
pluripotency. Aberrations in chromatin remodeling proteins are found to be associated with
human diseases, including cancer. Targeting chromatin remodeling pathways is currently
evolving as a major therapeutic strategy in the treatment of several cancers.
Access to nucleosomal DNA is governed by two major classes of protein complexes:
1) Covalent histone-modifying complexes.

2) ATP-dependent chromatin remodeling complexes.
Histone modifiers
Covalent histone-modifying complexes:

Specific protein complexes, known as histone-modifying complexes catalyze addition or

removal of various chemical elements on histones. These enzymatic modifications include
acetylation, methylation, phosphorylation, and ubiquitination and primarily occur at N-terminal
histone tails. Such modifications affect the binding affinity between histones and DNA, and thus
loosening or tightening the condensed DNA wrapped around histones, e.g., Methylation of
specific lysine residues in H3 and H4 causes further condensation of DNA around histones, and
thereby prevents binding of transcription factors to the DNA that lead to gene repression. On the
contrary, histone acetylation relaxes chromatin condensation and exposes DNA for TF binding,
leading to increased gene expression.
The histone code is a hypothesis that the transcription of genetic information encoded in DNA is
in part regulated by chemical modifications to histone proteins, primarily on their unstructured
ends. Together with similar modifications such as DNA methylation it is part of the epigenetic
code.
Known modifications
Well characterized modifications to histones include:
 Acetylation tends to define the ‗openness‘ of chromatin as acetylated histones cannot
pack as well together as deacetylated histones. Acetylation - by HAT (histone acetyl
transferase); deacetylation - by HDAC (histone deacetylase)
 Methylation
Both lysine and arginine residues are known to be methylated. Methylated lysines are the best
understood marks of the histone code, as specific methylated lysine match well with gene
expression states. Methylation of lysines H3K4 and H3K36 is correlated with transcriptional
activation while demethylation of H3K4 is correlated with silencing of the genomic region.
Methylation of lysines H3K9 and H3K27 is correlated with transcriptional repression.
Particularly, H3K9me3 is highly correlated with constitutive heterochromatin.
 Phosphorylation
 Ubiquitination
A very basic summary of the histone code for gene expression status is given below (histone
nomenclature is described in the Table below:
Table 1
Histone
Type of
modification
H3K4 H3K9 H3K14 H3K27 H3K79 H4K20 H2BK5
MONO-
activation activation activation activation activation activation
METHYLATION
DI-METHYLATION repression repression activation
TRI- Activation,
activation repression repression repression
METHYLATION repression
ACETYLATION activation activation
Fig.7 Histone modifications
Co-activators & Co-repressors
Enhancers:
Enhancers are DNA-regulatory elements that activate transcription of a gene or genes to higher
levels than would be the case in their absence. These elements function at a distance by forming
chromatin loops to bring the enhancer and target gene into proximity. An enhancer doesn't need
to be located near the transcription initiation site to affect transcription, as some have been found
located in several hundred thousand base pairs upstream or downstream of the start site.
Enhancers do not act on the promoter region itself, but are bound by activator proteins.
Enhancers v/s promoters:
An enhancer is a sequence of DNA that functions to enhance transcription. A promoter is a

sequence of DNA that initiates the process of transcription. A promoter has to be close to the
gene that is being transcribed while an enhancer does not need to be close to the gene of interest.
Repressors binding to enhancer ?
Like prokaryotic cells, eukaryotic cells also have mechanisms to prevent transcription.
Transcriptional repressors can bind to promoter or enhancer regions and block transcription. Like
the transcriptional activators, repressors respond to external stimuli to prevent the binding of
activating transcription factors.
RNA polymerase- structure and function of RNA polymerases (prokaryotes & eukaryotes)
RNAP was discovered independently by Charles Loe, Audrey Stevens, and Jerard Hurwitz in
1960. A RNA polymerase (RNAP), or ribonucleic acid polymerase, is a multi subunit enzyme
that catalyzes the process of transcription where an RNA polymer is synthesized from a DNA
template. The sequence of the RNA polymer is complementary to that of the template DNA and
is synthesized in a 5'→ 3′ orientation.
RNA polymerase (green) synthesizes RNA by following a strand of DNA. RNA polymerase is
an enzyme that is responsible for copying a DNA sequence into an RNA sequence, duyring the
process of transcription. RNA polymerases are enzymes that transcribe DNA into RNA. RNA
polymerase synthesizes an RNA strand complementary to a template DNA strand. It synthesizes
the RNA strand in the 5' to 3' direction, while reading the template DNA strand in the 3' to 5'
direction. The template DNA strand and RNA strand are antiparallel. RNA polymerases
transcribe the information in DNA into RNA molecules that have a variety of functions,
including messenger RNA (mRNA; codes for proteins), and non-coding RNAs such as transfer
RNA (tRNA; transports amino acids to the ribosome for protein synthesis), ribosomal RNA
(rRNA; helps catalyze protein synthesis.
All eukaryotes have three different RNA polymerases (RNAPs) which transcribe different types
of genes. RNA polymerase I transcribes rRNA genes, RNA polymerase II transcribes mRNA,
miRNA, snRNA, and snoRNA genes, and RNA polymerase III transcribes tRNA and 5S rRNA
genes.
Using the enzyme helicase, RNAP locally opens the double-stranded DNA so that one strand of
the exposed nucleotides can be used as a template for the synthesis of RNA, a process called
transcription. A transcription factor and its associated transcription mediator complex must be
attached to a DNA binding site called a promoter region before RNAP can initiate the DNA
unwinding at that position. RNAP not only initiates RNA transcription, it also guides the
nucleotides into position, facilitates attachment and elongation, has intrinsic proofreading and
replacement capabilities, and termination recognition capability. In eukaryotes, RNAP can build
chains as long as 2.4 million nucleotides.
RNAP produces RNA that, functionally, is either for protein coding, i.e. messenger RNA
(mRNA); or non-coding (so-called "RNA genes"). At least four functional types of RNA genes
exist:
1. transfer RNA (tRNA) — transfers specific amino acids to growing polypeptide chains at
the ribosomal site of protein synthesis during translation;
2. ribosomal RNA (rRNA) — incorporates into ribosomes;
3. micro RNA (miRNA) — regulates gene activity; and,
4. catalytic RNA (ribozyme) — functions as an enzymatically active RNA molecule.
RNA polymerase is essential to life, and is found in all living organisms and many viruses.
Depending on the organism, a RNA polymerase can be a protein complex (multi-subunit RNAP)
or only consist of one subunit (single-subunit RNAP, ssRNAP), each representing an
independent lineage. The former is found in bacteria, archaea, and eukaryotes alike, sharing a
similar core structure and mechanism. The latter is found in phages as well as eukaryotic
chloroplasts and mitochondria, and is related to modern DNA polymerases. Eukaryotic and
archaeal RNAPs have more subunits than bacterial ones do, and are controlled differently.
Bacteria and archaea only have one RNA polymerase. Eukaryotes have multiple types of nuclear
RNAP, each responsible for synthesis of a distinct subset of RNA:
 RNA polymerase I synthesizes a pre-rRNA 45S (35S in yeast), which matures and will
form the major RNA sections of the ribosome.
 RNA polymerase II synthesizes precursors of mRNAs and most sRNA and microRNAs.
 RNA polymerase III synthesizes tRNAs, rRNA 5S and other small RNAs found in the
nucleus and cytosol.
 RNA polymerase IV and V found in plants are less understood; they make siRNA. In
addition to the ssRNAPs, chloroplasts also encode and use a bacteria-like RNAP.
Function:
Control of the process of gene transcription affects patterns of gene expression and, thereby,
allows a cell to adapt to a changing environment, perform specialized roles within an organism,
and maintain basic metabolic processes necessary for survival. Therefore, it is hardly surprising
that the activity of RNAP is long, complex, and highly regulated.
 The function of RNA polymerase is to control the process of transcription, through which
copying of information stored in DNA into a new molecule of messenger RNA (mRNA.)
 During transcription, the RNA polymer is contemporary to the template DNA that is
synthesized in the direction of 5′ to 3′.
 The enzyme RNA polymerase interacts with proteins to enable it to function in catalyzation
of the synthesis of RNA.
 The collaborator proteins assist in enabling the specific binding of RNA polymerase, assist
in the unwinding of the double chemical structure of DNA, moderate the enzymatic
activities of RNA polymerase and to control the speed of transcription.
 The RNA polymerase enzyme has an interrupted mechanism whereby it continuously
synthesizes RNA polymers of over four thousand bases per minute but they pause or stop
occasionally to maintain fidelity.
 RNA polymerase is an enzyme that is responsible for copying a DNA sequence into an
RNA sequence, during the process of transcription. As a complex molecule composed of
protein subunits, RNA polymerase controls the process of transcription, during which the
information stored in a molecule of DNA is copied into a new molecule of messenger RNA.
 RNA polymerases have been found in all species, but the number and composition of these
proteins vary across taxa.
 For instance, bacteria contain a single type of RNA polymerase, while eukaryotes
(multicellular organisms and yeasts) contain three distinct types.
 In spite of these differences, there are striking similarities among transcriptional
mechanisms.
Products of RNAP include:
 Messenger RNA (mRNA)—template for the synthesis of proteins by ribosomes.

 Non-coding RNA or "RNA genes"—a broad class of genes that encode RNA that is not
translated into protein. The most prominent examples of RNA genes are transfer RNA
(tRNA) and ribosomal RNA (rRNA), both of which are involved in the process of
translation. However, since the late 1990s, many new RNA genes have been found, and
thus RNA genes may play a much more significant role than previously thought.
 Transfer RNA (tRNA)—transfers specific amino acids to growing polypeptide chains at
the ribosomal site of protein synthesis during translation
 Ribosomal RNA (rRNA)—a component of ribosomes
 Micro RNA—regulates gene activity
 Catalytic RNA (Ribozyme)—enzymatically active RNA molecules
RNAP accomplishes de novo synthesis. It is able to do this because specific interactions with the
initiating nucleotide hold RNAP rigidly in place, facilitating chemical attack on the incoming
nucleotide. Such specific interactions explain why RNAP prefers to start transcripts with ATP
(followed by GTP, UTP, and then CTP). In contrast to DNA polymerase, RNAP includes
helicase activity, therefore no separate enzyme is needed to unwind DNA.
Steps involved:
 Initiation:
RNA polymerase binding in bacteria involves the sigma factor recognizing the core promoter
region containing the −35 and −10 elements (located before the beginning of sequence to be
transcribed) and also, at some promoters, the α subunit C-terminal domain recognizing promoter
upstream elements. There are multiple interchangeable sigma factors, each of which recognizes a
distinct set of promoters. For example, in E. coli, σ70 is expressed under normal conditions and
recognizes promoters for genes required under normal conditions ("housekeeping genes"), while
σ32 recognizes promoters for genes required at high temperatures ("heat-shock genes"). In
archaea and eukaryotes, the functions of the bacterial general transcription factor sigma are
performed by multiple general transcription factors that work together. The RNA polymerase-
promoter closed complex is usually referred to as the "transcription preinitiation complex."
 Elongation:
The 17-bp transcriptional complex has an 8-bp DNA-RNA hybrid, that is, 8 base-pairs involve
the RNA transcript bound to the DNA template strand. As transcription progresses,
ribonucleotides are added to the 3' end of the RNA transcript and the RNAP complex moves
along the DNA. The characteristic elongation rates in prokaryotes and eukaryotes are about 10–
100 nts/sec.
 Termination:
In bacteria, termination of RNA transcription can be rho-dependent or rho-independent. The

former relies on the rho factor, which destablizes the DNA-RNA heteroduplex and causes RNA
release. The latter, also known as intrinsic termination, relies on a palindromic region of DNA.
Transcribing the region causes the formation of a "hairpin" structure from the RNA transcription
looping and binding upon itself. This hairpin structure is often rich in G-C base-pairs, making it
more stable than the DNA-RNA hybrid itself. As a result, the 8 bp DNA-RNA hybrid in the
transcription complex shifts to a 4 bp hybrid. These last 4 base pairs are weak A-U base pairs,
and the entire RNA transcript will fall off the DNA.
RNA polymerases in prokaryotes
In bacteria, the same enzyme catalyzes the synthesis of mRNA and non-coding RNA (ncRNA).
The prokaryotes have a single type of RNA polymerase (RNAP) which synthesizes all the
classes of RNA, i.e mRNA, tRNA, rRNA, sRNA. RNA Polymerase molecule is made up of 2
domains and 5 subunits:
i. Core and holoenzyme
ii. Subunits (β, β‘, α (αI and αII), ω,)
 The promoter is the sequence of DNA that is required for accurate and specific initiation of
transcription, and also, it is the sequence of DNA to which RNA polymerase binds
accurately to initiate transcription.
 The ‗a‘ subunit is made up of two distinct domains. The N-terminal domain (a-NTD) and
the C-terminal.
 The N-terminal is involved in dimerization forming a2 and further assembly of the RNA
polymerase.
 The C-terminal domain functions such as binding to the Upstream Promoter (UP) DNA
sequence at promoters for rRNA and tRNA genes and in communication with several
transcriptional activators.
RNAP is a large molecule. The core enzyme has five subunits (~400 kDa):
 β': The β' subunit is the largest subunit, and is encoded by the rpoC gene. The β' subunit
contains part of the active center responsible for RNA synthesis and contains some of the
determinants for non-sequence-specific interactions with DNA and nascent RNA. It is split
into two subunits in Cyanobacteria and chloroplasts.
 β: The β subunit is the second-largest subunit, and is encoded by the rpoB gene. The β
subunit contains the rest of the active center responsible for RNA synthesis and contains the
rest of the determinants for non-sequence-specific interactions with DNA and nascent RNA.
 α: The α subunit is the third-largest subunit and is present in two copies per molecule of
RNAP, αI and αII (one and two). Each α subunit contains two domains: αNTD (N-Terminal
domain) and αCTD (C-terminal domain). αNTD contains determinants for assembly of
RNAP. αCTD (C-terminal domain) contains determinants for interaction with promoter
DNA, making non-sequence-non-specific interactions at most promoters and sequence-
specific interactions at upstream-element-containing promoters, and contains determinants

for interactions with regulatory factors.
 ω: The ω subunit is the smallest subunit. The ω subunit facilitates assembly of RNAP and
stabilizes assembled RNAP.
In order to bind promoters, RNAP core associates with the transcription initiation
factor sigma (σ) to form RNA polymerase holoenzyme. Sigma reduces the affinity of RNAP for
nonspecific DNA while increasing specificity for promoters, allowing transcription to initiate at
correct sites. The complete holoenzyme therefore has 6 subunits: β'βαI and αIIωσ (~450 kDa).
RNA polymerases in eukaryotes
Eukaryotes have multiple types of nuclear RNAP, each responsible for synthesis of a distinct
subset of RNA. All are structurally and mechanistically related to each other and to bacterial
RNAP:
 RNA polymerase I synthesizes a pre-rRNA 45S (35S in yeast), which matures into 28S, 18S
and 5.8S rRNAs, which will form the major RNA sections of the ribosome.
 RNA polymerase II synthesizes precursors of mRNAs and
most snRNA and microRNAs. This is the most studied type, and, due to the high level of
control required over transcription, a range of transcription factors are required for its
binding to promoters.
 RNA polymerase III synthesizes tRNAs, rRNA 5S and other small RNAs found in
the nucleus and cytosol.
 RNA polymerase IV synthesizes siRNA in plants.
 RNA polymerase V synthesizes RNAs involved in siRNA-
directed heterochromatin formation in plants.
 RNA polymerase IV and V found in plants are not well understood, however, they make
siRNA. The plant chloroplast encodes the ssRNAPs and uses bacteria-like RNA Polymerase.
 Each of the nuclear RNA polymerases is a large protein molecule with about 8 to 14 subunits
and the molecular weight is approximately 500,000 for each.
 They commonly have 3 subunits, a, b and b‘. The largest subunits being b and b‘.
 These subunits are used as catalytic promoters and for assembly of proteins.
 Each of these polymerases has a different function:
RNA polymerase I
 This enzyme is located in the nucleolus of the cell.
 It is a specialized nuclear substructure where the ribosomal RNA (rRNA) is synthesized by
transcription and assembled into ribosomes.
 The rRNA are component elements of the ribosomes and are important in the process of
translation.
 Therefore, RNA polymerase I synthesize almost all rRNAs except 5S rRNA.

 In yeast, the enzyme has a mass of 600kDa and 13 subunits.
RNA polymerase II
 This enzyme is located in the nucleus.
 Most organisms that possess RNA polymerase II have a 12-subunit RNAP II (with a mass
of about 550 kDa)
 It is structurally made up of holoenzyme and mediators, with General Transcriptional
factors (GTFs).
 They contain transcription factors and transcriptional regulators.
 It functions by synthesizing all proteins that code for the nuclear pre-mRNAs in eukaryotic
cells (mRNAs in prokaryotic cells).
 It is responsible for transcribing most of the eukaryotic genes and especially found in
human genes.
RNA polymerase III
 It is located in the nucleus.
 The RNA polymerase III has 14 or more distinct subunits with a mass of approximately 700
kDa.
 Its function is to transcribe transfer RNA (tRNA), ribosomal RNA (rRNA), and other small
RNAs.
 Some of its target points are important for the normal functioning of the cell
RNA polymerases IV and V
 They are exclusively found in plants, and they perform combined action in the formation of
small interfering RNA and heterochromatin in the cell nucleus.
 In Plants, the RNA polymerase is found in the chloroplast (plastids) and mitochondria,
encoded by the mitochondrial DNA.
 These enzymes are much more related to bacterial RNA polymerase than to the nuclear
RNA polymerase.
 Their function is to catalyze specific transcription of organelle genes.
Functions of RNA polymerases:
 Generally, the RNA molecule is a messenger molecule that is used to export information
that is coded in DNA out of the cell nucleus, to synthesize proteins in the cell cytoplasm.
 RNA polymerase is used in the production of molecules that play a wide range of roles, of
which one of its functions is to regulate the number and type of RNA transcript that is
formed in response to the requirements of the cell.
 The RNA polymerase enzyme interacts with different molecular proteins, transcription
factors, and signaling molecules on the carboxyl-terminal, which regulates its mechanisms,
which play a major role in gene expression and gene specialization in multicellular
(eukaryotic) organisms.
 The RNA enzyme also ensures irregularities and errors during the conversion of DNA to
RNA (transcription). Such as ensuring that the right nucleotide is added to the newly
synthesized RNA strand, inserting the right amino acid-base which is complementary to the
template of the DNA strand.
 When the right nucleotides have been inserted, the RNA polymerase can then catalyze and
elongate the RNA strand, at the same time, proofread the new strand and remove incorrect
bases.
 RNA polymerase is also involved in the post-transcription modification of RNAs,
converting them into functional molecules that facilitate the transportation of molecules
from the nucleus to their site of action.
 Besides its role in the synthesis of proteins, RNA performs other functions such as
 Protein coding
 Regulation of gene expression
 Act as enzymes
 Formation of gametes by the non-coding RNA (ncRNA)
 Production of regulatory molecules.
Mechanism of transcription in prokaryotes and eukaryotes
Transcription is the process by which the information in a strand of DNA is copied into a new
molecule of messenger RNA (mRNA). The newly formed mRNA copies of the gene then serve
as blueprints for protein synthesis during the process of translation. With the genes bound in the
nucleus, transcription occurs in the nucleus of the cell and the mRNA transcript must be
transported to the cytoplasm. There is no such structure seen in prokaryotes. Another main
difference between the two is that transcription and translation occurs simultaneously in
prokaryotes and in eukaryotes the RNA is first transcribed in the nucleus and then translated in
the cytoplasm. Eukaryotes contain mRNAs that are monocystronic.
Prokaryotic transcription
In prokaryotes, which lack membrane-bound nuclei and other organelles, transcription occurs in
the cytoplasm of the cell.
Transcription involves four steps:

 Initiation. The DNA molecule unwinds and separates to form a small open complex.
 Elongation. RNA polymerase moves along the template strand, synthesising an mRNA molecule.
 Termination. In prokaryotes there are two ways in which transcription is terminated.
 Processing.
Well, apart from being proteins to control transcription in Prokaryotes, they are homologous to
archaeal transcription factor B and to eukaryotic factor TFIIB. Sigma factors are also needed at
the promoter to initiate transcription, while transcription factors regulate the gene expression.
RNA Polymerase is the enzyme that produces the mRNA molecule (just like DNA polymerase
produced a new DNA molecule during DNA replication). Prokaryotes use the same RNA
polymerase to transcribe all of their genes. In E. coli, the polymerase is composed of five
polypeptide subunits. These subunits assemble every time a gene is transcribed, and they
disassemble once transcription is complete. Each subunit has a unique role (which you do not
need to memorize). The polymerase comprised of all five subunits is called the holoenzyme.
Initiation:
Transcription in prokaryotes (and in eukaryotes) requires the DNA double helix to partially
unwind in the region of mRNA synthesis. The region of unwinding is called a transcription
bubble. The DNA sequence onto which the proteins and enzymes involved in transcription bind
to initiate the process is called a promoter. In most cases, promoters exist upstream of the genes
they regulate. The specific sequence of a promoter is very important because it determines
whether the corresponding gene is transcribed all of the time, some of the time, or hardly at all.
The structure and function of a prokaryotic promoter is relatively simple (Figure 1). One
important sequence in the prokaryotic promoter is located 10 bases before the transcription start
site (-10) and is commonly called the TATA box.
Fig 8 : The general structure of a prokaryotic promoter
To begin transcription, the RNA polymerase holoenzyme assembles at the promoter. The
dissociation of σ allows the core enzyme to proceed along the DNA template, synthesizing
mRNA by adding RNA nucleotides according to the base pairing rules, similar to the way a new
DNA molecule is produced during DNA replication. Only one of the two DNA strands is
transcribed. The transcribed strand of DNA is called the template strand because it is the
template for mRNA production. The mRNA product is complementary to the template strand
and is almost identical to the other DNA strand, called the non-template strand, with the
exception that RNA contains a uracil (U) in place of the thymine (T) found in DNA. Like DNA
polymerase, RNA polymerase adds new nucleotides onto the 3′-OH group of the previous
nucleotide. This means that the growing mRNA strand is being synthesized in the 5′ to 3′
direction. Because DNA is anti-parallel, this means that the RNA polymerase is moving in the 3′
to 5′ direction down the template strand.
Elongation:
As elongation proceeds, the DNA is continuously unwound ahead of the core enzyme as the
hydrogen bonds that connect the complementary base pairs in the DNA double helix are broken.
The DNA is rewound behind the core enzyme as the hydrogen bonds are reformed. The base
pairing between DNA and RNA is not stable enough to maintain the stability of the mRNA
synthesis components. Instead, the RNA polymerase acts as a stable linker between the DNA
template and the newly forming RNA strand to ensure that elongation is not interrupted
prematurely.
Fig 9 : During elongation, RNA polymerase tracks along the DNA template, synthesizes
mRNA in the 5′ to 3′ direction, and unwinds then rewinds the DNA as it is read
Termination:
Once a gene is transcribed, the RNA polymerase needs to be instructed to dissociate from the
DNA template and liberate the newly made mRNA. Depending on the gene being transcribed,
there are two kinds of termination signals. One is protein-based and the other is RNA-based.
Both termination signals rely on specific sequences of DNA near the end of the gene that cause
the polymerase to release the mRNA.
In a prokaryotic cell, by the time transcription ends, the transcript would already have been used
to begin making copies of the encoded protein because the processes of transcription and
translation can occur at the same time since both occur in the cytoplasm. In contrast,
transcription and translation cannot occur simultaneously in eukaryotic cells since transcription
occurs inside the nucleus and translation occurs outside in the cytoplasm.
Fig 10 : Multiple polymerases can transcribe a single bacterial gene while numerous
ribosomes concurrently translate the mRNA transcripts into polypeptides. In this way, a
specific protein can rapidly reach a high concentration in the bacterial cell.
Eukaryotic transcription
In eukaryotes, transcription and translation take place in different cellular compartments:

transcription takes place in the membrane-bounded nucleus, whereas translation takes place
outside the nucleus in the cytoplasm. In prokaryotes, the two processes are closely coupled. In
eukaryotes, transcription factors (like most proteins) are transcribed in the nucleus but are then
translated in the cell's cytoplasm. Many proteins that are active in the nucleus contain nuclear
localization signals that direct them to the nucleus.
Fig.11 RNA processing
Initiation:
The TATA box-binding protein of TFIID bends the TATA box DNA allowing the TFIIBC(C-
terminal) binds sequences up and downstream of the TATA box. This causes the DNA to curve
and wrap around the RNA polymerase II. The TFIIBN (N-terminal) binds to the dock domain,
saddle, and active center cleft of RNA polymerase II. When TFIIB binds the promoter DNA is
directed toward and above the active center of RNA polymerase II, running across the face of the
initiation complex, passing the Tfg2 subunit of TFIIF above the cleft passing TFIIE and TFIIH at
the downstream end of the cleft. The path of the DNA seems to be completely determined by
interactions with transcription factors. It is believed that the DNA does not interact with the
RNA polymerase II before the DNA is separated into single strands.
TFIIE interacts with the RNA polymerase II jaws. TFIIH, which is bound to TFIIE, has helicase
activity and causes a quick promoter opening. This open region of the non-template DNA is
stabilized by the subunit Tfg2 of TFIIF. The template strand is stabilized by the TFIIB finger
and RNA polymerase in the active center cleft. This allows the DNA to become more flexible
because it is single stranded. It is at this point that the DNA first bends and proceeds into the
cleft. The DNA then first interacts with RNA polymerase II in the active center and with what
will become the transcription bubble.
The nucleoside triphosphates must be held in positions +1 and -1 for the synthesis of the first
phosphodiester bond. After the first few are added the nucleotides must still be held by protein-
RNA interactions. Sometimes these interactions are not sufficient to hold and the nucleotides are
released. This means the RNA polymerase must start again. This is referred to as ―abrortive
cycling‖. The TFIIB finger is only stabilizes a RNA/DNA hybrid for up to 9 base pairs after 9
base pairs the TFIIBN competes to occupy the RNA polymerase II saddle. This is what is
believed to account for the fact that TFIIB dissociates from the complex. Before TFIIB
dissociates TFIIH phosphorylates the CTD domain of Rpb1. This is the end of initiation.
Order of transcription factor binding:
TFIID binds to the TATA box
TFIIB binds to TFIID
TFIIF and RNA polymerase II binds to TFIIB and TFIID
TFIIH binds completing the initiation complex
Elongation:
Active Site: The active site is described as area of nucleotide addition. It contains two
Mg2+ ions named A and B. Metal A is bound is bound by the aspartates D481, D483, and D485
of Rpb1. Metal B is coordinated by Rpb1 residues D481, D483, and Rpb2 residue D836.
Transcription Bubble: The downstream DNA lies in the cleft between the clamp and
the subunit Rpb2. The DNA contacts the ―lower jaw‖ of subunit Rpb5 and then passes between
the Rpb2 ―lobe‖ and the Rpb1 ―clamp head‖. The downstream portion of the transcription
bubble starts where the DNA begins to unwind, which is approximately three to four nucleotides
before the RNA/DNA hybrid. The template strand proceeds along the bottom of the clamp over
the helical ―bridge‖. The +1 nucleotide is flipped compared to the +2 nucleotide by a left-
handed twist of 90o. This means that the +1 nucleotide points to the floor of the cleft and the
active site. Residues Ala832 and Thr831 position the +1 coding nucleotide and Tyr836 binds to
nucleotide +2 to stabilize them. The stabilization of the downstream portion of the transcription
bubble can also be due to the binding to fork loop 2 of Rpb2. The fork loop 1 of Rpb1 may also
help stabilize the transcription bubble further upstream.
RNA Synthesis:
NTPs for transcription enter through the pore, which is found in Rpb1 in the floor of the cleft
beneath the active site above the +1 site. When the NTP enters the RNA polymerase II it brings
with it metal B. The NTP and B metal ion enter the E site (10). If the NTP is the correct
nucleotide the nucleotide is then rotated into the A site where the A metal coordinates the alpha
phosphate of the NTP along with the aspartate residues 481, 483, and 485 from Rpb1. The B
metal is now coordinated by the beta and the gamma phosphates of the NTP as well as the
aspartate residues 481 and 483 in Rpb1 and the asp residue 836 from Rpb2. The nucleotide that
was positioned at the +1 site has vander Walls interactions with Thr831 and Ala832 which are in
the ―bridge‖ that was described above on the Rpb1 subunit. This helical bridge oscillates
between a straight and bent conformation. When the nucleotide is added at the +1 position would
be straight. When the bridge bends it translocates the nucleotide in the +1 position into the -1
position opening up the +1 site for another nucleotide to be added. If the NTP is not the correct
nucleotide it will stay in the E site and not rotate into the A site.
Separation of RNA from DNA and RNA Exit:
The separation of RNA from the DNA/RNA hybrid begins at position -8 with the residues after
that being completely separated. Three protein loops interact with the RNA/DNA hybrid to
assist in separation: the ―lid‖, ―rudder‖, and ―fork loop 1‖. The lid, which is composed of
residues 246-264 from Rpb1, interacts with residues -8, -9, and -10 and acts as a wedge to break
the RNA from the DNA. It also maintains the separation and guides the RNA to the exit path.
The lid interacts with other proteins to form an arch The plane of the aromatic side chain of
Phe252 interacts with the hybrid and splits it. The rudder, which is composed of residues 310-
324 from the subunit Rpb1, prevents reassociation of the complex by interacting with the DNA
at -9 and -10. This is accomplished by contact with the Ser318 and Arg320 at the 5‘ phosphate
at the -10 position. Fork loop 1, which is composed of residues 461-480 of Rpb2, is believed to
prevent unwinding of the hybrid past position -8. The residues Lys471 and Arg476 appear to
contact the RNA phosphates at positions -5, -6, and -7. These three loops lie near the saddle,
which is underneath the arch, and between the clamp and wall.
The RNA exits through the so-called exit pore, which is the area beneath the arch. After the arch
the RNA follows one of two proposed exit paths. In the first path the RNA follows a positively
charged grove that runs around the base of the clamp and towards the Rpb7 subunit, which has
the ability to bind single stranded RNA. In the second path a positively charged groove that runs
down the back of RNA polymerase II leads to the Rpb8 subunit, which could also interact with
single stranded RNA. The second path is favored because the RNA would have to take a sharp
bend after passing beneath the lid if the first path was plausible.
Termination:
There is not much known about the process of termination in eukaryotes, but it is known that it is
dependent upon poly(A) signals and downstream terminator sequences. The details of this
process are not very well known and are probably very variable. The post-transcriptional
modifications of the RNA occur at the same time as transcription. The post-transcriptional
modifications occur at the 5‘ and the 3‘ ends of the RNA molecule. The 5‘ end undergoes
capping. The first step is when the phosphoryl group is released by hydrolysis. The diphosphate
5‘ end then attacks the alpha phosphorus atom of GTP. This forms a 5‘-5‘ triphosphate linkage
that protects it from degradation from 5‘ endonucleases. This is referred to as the cap. When the
N-7 nitrogen of the terminal guanine is methylated it is referred to as cap 0. When the other
ribosomes are methylated it is referred to as cap 1 and cap 2. The 3‘ end contains a
polyadenylated tail. The RNA transcripts are cleaved by an endonuclease that recognizes the
signal AAUAAA, which is part of the cleavage signal. A poly(A) polymerase adds about 250
adenylate residues to the 3‘ end of the transcript. Another event in posttranscriptional processing
is splicing. In which the introns are removed from the RNA and the exons are then linked to
form the mature mRNA.
Post-transcriptional processing (RNA editing, splicing, poly A tail and 5’ capping, siRNA,
miRNAs, other ncRNAs)
Post-transcriptional modification or co-transcriptional modification is a set of biological

processes common to most eukaryotic cells by which an RNA primary transcript is chemically
altered following transcription from a gene to produce a mature, functional RNA molecule that
can then leave the nucleus and move to cytoplasm for further translation process.
The three processes that make up these post- transcriptional modifications:
RNA editing
RNA editing (also RNA modification) is a molecular process through which some cells can
make discrete changes to specific nucleotide sequences within an RNA molecule after it has been
generated by RNA polymerase. It occurs in all living organisms, and is one of the most
evolutionarily conserved properties of RNAs. RNA editing is widely observed in eukaryotic
organisms and their viruses. Editing, like splicing, represents a form of processing that has the
capacity to amplify genetic diversity and alter gene product function by modifying the
information transfer process at the posttranscriptional level.
Fig.12 RNA splicing
RNA splicing, in molecular biology, is a form of RNA processing in which a newly made
precursor messenger RNA (pre-mRNA) transcript is transformed into a mature messenger RNA
(mRNA). During splicing, introns (non-coding regions) are removed and exons (coding regions)
are joined together.
5' capping, addition of the poly A tail, and splicing.
The 5' capping reaction replaces the triphosphate group at the 5' end of the RNA chain with a
special nucleotide that is referred to as the 5' cap. In this step, 7-methylguanosine is added to 5'
end of RNA. During Poly-adenylation step, poly A tail is added to 3' end of RNA.
siRNA
Small interfering RNA (siRNA), sometimes known as short interfering RNA or silencing RNA,
is a class of double-stranded RNA non-coding RNA molecules, typically 20-27 base pairs in
length, similar to miRNA, and operating within the RNA interference (RNAi) pathway. The
main function of siRNA is to protect the cell from the exogenous mRNA attacks.
Functionally, the siRNA degrades the growing mRNA (exogenous as well as endogenous) and
stop gene expression. The origin of the siRNA is exogeneous, it came from the viral infections.
During RNAi, long dsRNA is cut or "diced" into small fragments ~21 nucleotides long by an
enzyme called "Dicer". These small fragments, referred to as small interfering RNAs (siRNA),
bind to proteins from a special family: the Argonaute proteins.
Fig. 13 Sliced mRNA
miRNAs
A microRNA (abbreviated miRNA) is a small non-coding RNA molecule (containing about 22

nucleotides) found in plants, animals and some viruses, that functions in RNA silencing and
post-transcriptional regulation of gene expression. miRNAs function via base-pairing with
complementary sequences within mRNA molecules. The miRNA functions as a guide by base-
pairing with target mRNA to negatively regulate its expression. The level of complementarity
between the guide and mRNA target determines which silencing mechanism will be employed;
cleavage of target messenger RNA (mRNA) with subsequent degradation or translation
inhibition. They generally bind to the 3'-UTR (untranslated region) of their target mRNAs and
repress protein production by destabilizing the mRNA and translational silencing.
There are now over 2000 miRNAs that have been discovered in humans and it is believed that
they collectively regulate one third of the genes in the genome. miRNAs have been linked to
many human diseases and are being pursued as clinical diagnostics and as therapeutic targets.
Fig. 14 mRNA processing steps in nucleus and cytosol
ncRNAs
A non-coding RNA (ncRNA) is a functional RNA molecule that is transcribed from DNA but
not translated into proteins. The DNA sequence from which a functional non-coding RNA is
transcribed is often called an RNA gene. Epigenetic related ncRNAs include miRNA, siRNA,
piRNA and lncRNA. In general, ncRNAs function to regulate gene expression at the
transcriptional and post-transcriptional level. The number of non-coding RNAs within the human
genome is unknown; however, recent transcriptomic and bioinformatic studies suggest that there
are thousands of them. Many of the newly identified ncRNAs have not been validated for their
function. It is also likely that many ncRNAs are non functional (sometimes referred to as junk
RNA), and are the product of spurious transcription.
Fig. 15 Achieving nascent protein
In eukaryotes the spliceosome performs the splicing reactions essential for removing intron
sequences, this process is required for the formation of mature mRNA. The spliceosome is
another RNP often also known as the snRNP or tri-snRNP. There are two different forms of the
spliceosome, the major and minor forms. The ncRNA components of the major spliceosome are
U1, U2, U4, U5, and U6. The ncRNA components of the minor spliceosome are U11, U12, U5,
U4atac and U6atac. Another group of introns can catalyse their own removal from host
transcripts; these are called self-splicing RNAs. There are two main groups of self-splicing
RNAs: group I catalytic intron and group II catalytic intron. These ncRNAs catalyze their own
excision from mRNA, tRNA and rRNA precursors in a wide range of organisms.
Transcription inhibitors
Gene-specific inhibition of transcription can be accomplished by antisense RNA, triple-helix

formation and DNA-binding polyamides. The presence of the RNA polymerase is essential for
the binding.
Table 2: Transcription inhibitors
Regulation of transcription in prokaryotes
Much of the early understanding of transcription came from bacteria,.although the extent and
complexity of transcriptional regulation is greater in eukaryotes. Prokaryotic transcription and
translation occur simultaneously in the cytoplasm, and regulation occurs at the transcriptional
level. Bacterial transcription is governed by three main sequence elements:
 Promoters are elements of DNA that may bind RNA polymerase and other proteins for the
successful initiation of transcription directly upstream of the gene.
 Operators recognize repressor proteins that bind to a stretch of DNA and inhibit the
transcription of the gene.
 Positive control elements that bind to DNA and incite higher levels of transcription.
While these means of transcriptional regulation also exist in eukaryotes, the transcriptional
landscape is significantly more complicated both by the number of proteins involved as well as
by the presence of introns and the packaging of DNA into histones.
The transcription of a basic bacterial gene is dependent on the strength of its promoter and the
presence of activators or repressors. In the absence of other regulatory elements, a promoter's
sequence-based affinity for RNA polymerases varies, which results in the production of different
amounts of transcript. The variable affinity of RNA polymerase for different promoter sequences
is related to regions of consensus sequence upstream of the transcription start site.
In the absence of other regulatory elements, the default state of a bacterial transcript is to be in
the ―on‖ configuration, resulting in the production of some amount of transcript. This means that
transcriptional regulation in the form of protein repressors and positive control elements can
either increase or decrease transcription. Repressors often physically occupy the promoter
location, occluding RNA polymerase from binding. Alternatively a repressor and polymerase
may bind to the DNA at the same time with a physical interaction between the repressor
preventing the opening of the DNA for access to the minus strand for transcription.
There are two majors kinds of proteins that control prokaryotic transcription: repressors and
activators. Repressors bind to an operator region to block the action of RNA polymerase. The lac
operon is activated by the CAP (catabolite activator protein), which binds to the promoter to
stabilize RNA polymerase binding.
Fig.16 Gene regulation in eukaryotes
Regulation of transcription in eukaryotes
Eukaryotic gene expression is regulated during transcription and RNA processing, which take
place in the nucleus, and during protein translation, which takes place in the cytoplasm. The
added complexity of generating a eukaryotic cell carries with it an increase in the complexity of
transcriptional regulation. Eukaryotes have three RNA polymerases, known as Pol I, Pol II,
and Pol III. Each polymerase has specific targets and activities, and is regulated by independent
mechanisms. There are a number of additional mechanisms through which polymerase activity
can be controlled. These mechanisms can be generally grouped into three main areas:
 Control over polymerase access to the gene. This is perhaps the broadest of the three control
mechanisms. This includes the functions of histone remodeling enzymes, transcription
factors, enhancers and repressors, and many other complexes
 Productive elongation of the RNA transcript. Once polymerase is bound to a promoter, it
requires another set of factors to allow it to escape the promoter complex and begin
successfully transcribing RNA.
 Termination of the polymerase. A number of factors which have been found to control how
and when termination occurs, which will dictate the fate of the RNA transcript.
All three of these systems work in concert to integrate signals from the cell and change the
transcriptional program accordingly. As in bacteria, transcription in eukaryotic cells is controlled
by proteins that bind to specific regulatory sequences and modulate the activity of RNA
polymerase.
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DAYANANDA SAGAR COLLEGE OF ENGINEERING

Basal transcription complex formation

Eukaryotic transcriptional activators

Chromatin structure & regulation

Co-activators & Co-repressors

RNA polymerase- structure and function of RNA polymerases (prokaryotes &

Mechanism of transcription in prokaryotes and eukaryotes

Post-transcriptional processing (RNA editing, splicing, poly A tail and 5‘ capping,

Regulation of transcription in prokaryotes and eukaryotes

In molecular biology, a transcription factor (TF) (or sequence-specific DNA-binding factor) is a

There are two mechanistic classes of transcription factors:

Basal transcription complex formation

Eukaryotic transcriptional activators

Transcription of eukaryotic structural genes is regulated by promoter-specific activator proteins

Fig. 3 Binding sites

Chromatin structure & regulation

Fig.4 Epigenetic regulation of chromatin structure

Fig. 5 DNA packaging: Nucleosomes and chromatin

Chromatin remodeling is the rearrangement of chromatin from a condensed state to a

Fig.6 Gene switching on and off modes

Such remodeling is principally carried out by:

1) covalent histone modifications by specific enzymes, e.g., histone acetyltransferases (HATs),

2) ATP-dependent chromatin remodeling complexes which either move, eject or restructure

Access to nucleosomal DNA is governed by two major classes of protein complexes:

1) Covalent histone-modifying complexes.

Covalent histone-modifying complexes:

Specific protein complexes, known as histone-modifying complexes catalyze addition or

DI-METHYLATION repression repression activation

ACETYLATION activation activation

Fig.7 Histone modifications

Co-activators & Co-repressors

Enhancers v/s promoters:

An enhancer is a sequence of DNA that functions to enhance transcription. A promoter is a

Repressors binding to enhancer ?

Products of RNAP include:

 Messenger RNA (mRNA)—template for the synthesis of proteins by ribosomes.

In bacteria, termination of RNA transcription can be rho-dependent or rho-independent. The

RNA polymerases in prokaryotes

specific interactions at upstream-element-containing promoters, and contains determinants

RNA polymerases in eukaryotes

 Therefore, RNA polymerase I synthesize almost all rRNAs except 5S rRNA.

Functions of RNA polymerases:

Mechanism of transcription in prokaryotes and eukaryotes

Transcription involves four steps:

Fig 8 : The general structure of a prokaryotic promoter

In eukaryotes, transcription and translation take place in different cellular compartments:

Fig.11 RNA processing

Order of transcription factor binding:

TFIID binds to the TATA box

TFIIB binds to TFIID

TFIIF and RNA polymerase II binds to TFIIB and TFIID

TFIIH binds completing the initiation complex

Separation of RNA from DNA and RNA Exit:

Post-transcriptional modification or co-transcriptional modification is a set of biological

The three processes that make up these post- transcriptional modifications:

Fig.12 RNA splicing

5' capping, addition of the poly A tail, and splicing.

Fig. 13 Sliced mRNA

A microRNA (abbreviated miRNA) is a small non-coding RNA molecule (containing about 22

Fig. 14 mRNA processing steps in nucleus and cytosol

Fig. 15 Achieving nascent protein

Gene-specific inhibition of transcription can be accomplished by antisense RNA, triple-helix

Table 2: Transcription inhibitors