Fig. 11-CO, p.

264
 Learning Objectives

1. How Does Transcription Take Place in Prokaryotes?

2. How Is Transcription Regulated in Prokaryotes?
3. How Does Transcription Take Place in Eukaryotes?
4. How Is Transcription Regulated in Eukaryotes?
5. How Is RNA Modified after Transcription?
6. How Does RNA Act as an Enzyme?
 Gene expression
It is the action of a gene to produce single
polypeptide, which occurs in two steps:
Transcription of the information encoded in
single gene of chromosomal DNA into a
molecule of RNA.
Translation of the information encoded in the
nucleotides of mRNA into defined sequence of
amino acids in a polypeptide
 RNA transcription

Transcription produces RNA molecules that are

complimentary copies to one strand in DNA molecule.
The transcription of each gene starts
from specific sequence on DNA called
the promoter and finishes on another
sequence called the terminator. Both the
promoter and terminator are regulatory
regions separated from the coding
sequence of the DNA
Unlike DNA replication, the transcription is
unidirectional and only one of the two double
DNA strands act as a template. Since, the
Synthesis of RNA proceeds in the 5' 3'
direction, therefore in each transcription step
only the DNA strand having the 3 5'
direction is copied as a template. The copying
process involves small part but not the whole
DNA molecule.
Table 11-1, p.265
The basics of transcription

Fig. 11-1, p.265

 Prokaryotic RNA transcription
Transcription in prokaryotes is carried out by a
single RNA polymerase and performed in 3 stages:
initiation, elongation and termination.
The promoter region contains two boxes of
specific sequences located -10 bp and -35 bp
upstream (left side) of position +1, which
represents the first nucleotide on DNA strand
to be copied as a template.
With the help of sigma factor binding, the
RNA polymerase can strongly interact with
these two promoter boxes and produce large
amounts of RNA. Any binding of RNA
polymerase at promoter regions other than
these two specific boxes will be weak and
produces little RNA.
The most common nucleotide sequence
found in these two boxes are TATAAT at -10
region and TTGACA at -35 region
Sequence of representative promoters from E.coli

E. coli have 7 different sigma factors

B. subtilis have 18 different sigma factors
Fig. 11-2, p.266
The binding of sigma-RNA polymerase complex at
the -35 box also cause unwinding of short DNA
segment extended between -10 to +1 bp sequence to
make enough space for the initiation of transcription
process.
In this process the interacting complex undergoes a
conformational shift that leads to unwinding of the
double helix.
 Elongation
The open complex begins RNA synthesis
complementary to one of the strands of DNA
called sense strand, the opposite DNA strand
is called antisense strand. At this point sigma
factor dissociates from the RNA polymerase
enzyme which can be recycled again for
continued initiation of transcription.
During elongation of the RNA molecule, ribonucleoside
triphosphates of the bases A, U, G, and C pair with
complementary bases in the sense or template strand
of DNA and are then connected with 3'5'
phosphodiester bonds by RNA polymerase. Thus, the
RNA molecule grows from its 5' end toward its 3' end
(5' 3'). The DNA double helix unwinds ahead of the
advancing RNA polymerase to expose more of the
sense strand for extending the RNA chain. The DNA
reforms its double-helical shape after the enzyme has
passed a given region
Transcription occurs at 30-50 nucleotides/second
adding ribonucleotides monophosphate (supplied as
triphosphates ) in to RNA chain using the antisense
strand as a template. As each nucleoside triphosphate
is brought to the 3' end of the growing strand, the two
terminal phosphates are removed as pyrophosphates
similar to the reaction catalyzed by DNA polymerase.
The addition proceeds in the 5' 3' direction and
follow base pair ruling taking in consideration that
each A on the DNA guides the insertion of the
pyrimidine uracil (U) instead of T. The proofreading by
the enzyme insures the correct insertion of bases.
Sequence of events in the initiation &
elongation phases of transcription in prokaryotes

Fig. 11-3, p.268

 Termination
In bacteria transcription termination occurs by two
general mechanisms.
The first mechanism is called Rho dependant. The
Rho is a protein - a termination factor (similar to
sigma initiation factor) which recognizes a specific
DNA sequence and interacts with the transcription
machinery to terminate transcription .At that site the
Rho binds to separate the new RNA from Polymerase
and DNA template.
The second mechanism is called Rho independent
because it acts independently of Rho factor
The -independent terminators have a dyad symmetry
in the double-stranded DNA, centered about 1520
nucleotides before the end of the RNA, and have about
six adenines in the sense strand that are transcribed
into uracils at the end of the RNA.
dyad symmetry are two areas of a DNA strand whose
base pair sequences are inverted repeats of each other
such as the sequences GAATAC and GTATTC which
are reverse complements of each other.
The RNA transcript of the dyad symmetry folds back on
itself to form a hairpin structure, ending with
approximately 6 uracils .Because an RNA-DNA hybrid
consisting of polyribo-U and polydeoxyribo-A is very
unstable, the RNA chain is quickly released from the
DNA duplex.
Inverted repeats terminate transcription

Fig. 11-5, p.269

The rho(p)factor mechanism of transcription termination

Fig. 11-6, p.270

 Eukaryotic RNA transcription
Transcription in eukaryotes differs from the process in
prokaryotes by the following properties:
1. Genes are transcribed individually instead of groups.
2. Transcription occurs in a separate compartment (the
nucleus) from translation, which occurs in the cytoplasm
3. Initially transcription results in a pre-messenger RNA
(pre-mRNA) molecule that must be processed before it
emerges as a mature mRNA ready for translation
4. DNA is complexed with many proteins and is highly
compacted, and therefore must be unwound to expose
its promoters. Only genes occurring in regions of the
relaxed chromatin (unpacked) are prepared for
potential transcription .
Most transcriptionally inactive segment of DNA are found in the
highly condensed heterochromatin. Interconversion of the two
forms is called chromatin remodeling.
Two major factors influence chromosome structure. They are
DNA methylation and histone acetylation. Genes with more
methylation are less active in transcription,
while more acetylated histones produce looser chromatins,
which become more active.

Relaxed chromatin Highly packed chromatin

Genomic sequences packaged into Genomic sequences

free nucleosomes are accessible to organized into solenoid
transcription configuration are
inaccessible to
transcription machinery
5. Eukaryotes have three separate RNA polymerases
instead of one , each transcribing certain type of RNA.
RNA Polymerase I rRNAs (ribosomal RNA)
RNA Polymerase II mRNA precursors or
heterogenous RNA(hn-RNA)
RNA Polymerase III tRNA (transfer RNA) and
other small RNA
RNA polymerase II structure is similar to the core
structure of prokaryotic polymerase, but it has a larger
protein size with about 8 to 14 subunits instead of 4.
The largest subunit of RNA polymerase II has a
carboxy-terminal domain and called CTD. This
subunit can be phosphorylated by certain kinase
6. Unlike prokaryotes, RNA polymerase II is
unable to bind directly the promoter region
because it has no similar sigma subunit of
their prokaryotic . Instead, eukaryotic
polymerases depend on other proteins that
bind to the promoter regions and then bring
the RNA polymerases to the correct promoter
site.
 Promoter region of polymerase
II
Unlike prokaryotic promoters, eukaryotic
promoter regions do not have a fixed sequence
for starting the initiation of transcription. The
most common promoter sequence as initiator
element is "TATA box", with the sequence
TATAAAA . This region approximately 25 base
pairs upstream from the starting nucleotide and
almost similar to the -10 region found in
prokaryotic promoters.
Other non-universal sequences include the
"CAAT box" (GGCCAATCT) and the "GC box"
(GGGCG) located in promoter regions more
than 100 base pairs distance from the starting
transcription point. Each of these sequences
can act as binding sites for specific transcription
factors to assist in the binding of RNA
polymerases.
However, the presence of these three boxes
together is more common than their individual
presence in the promoter region. TATA Box By
Itself Makes for Only a Weak Promoter
 Additional promoter sequences are needed for a
strong promoter OR for regulated promoters that
provide binding sites for Gene-specific Transcription

In eukaryotes, the specific promoter sequence for a

gene transcribed by pol II is called core promoter
and most often found immediately upstream (5) of
the start site for the gene
 General transcription factors for
eukaryotic RNA polymerase II

The transcription factors are proteins that

control the activity of RNA pol II and because
they are required for all mRNA genes they are
called general transcription factors.
TFII means general transcription factor of
RNA pol II
RNA polymerase II requires Six General TFs:
TFIIA ,TFIIB ,TFIID ,TFIIE ,TFIIF ,TFIIH
. TFIID is transcription factor D for RNA polymerase II
. TFIID which is the key factor to Promoter Recognition
TFIID itself is a multi-protein complex composed of
TATA box binding protein (TBP) + the protein TATA-
associated factors (TAF).TBP plays a role in directing
RNA polymerase to initiate at the correct place
Initiation of transcription:
General transcription factors and the polymerase undergo a pattern of
sequential binding to initiate transcription of nuclear genes.
Formation of pre-initiation Complex
The TFIID interacts with the TATA box sequence of the DNA through the
TBP component to form TFs (transcription factors) complex. This initial
binding event creates binding sites for TFIIA and TFIIB to be formed.
TFIIF already bound to RNA pol II which can bring it to bind the TFs
complex at the TATA box region.
Further addition of TFIIE and TFIIH
completes the formation of pre- initiation
complex . The transcription factors bind to
DNA in a preferred order:
TFIID, then TFIIA, then TFIIB, then RNA Pol
II + TFIIF, then TFIIE
The template DNA is still a duplex and the next step in
the initiation process is the unwinding of the duplex
DNA at the start site for transcription.
Once Preinitiation Complex Forms, TFIIH binding will
activate the polymerase clearance from the promoter
region to start elongation.
TFIIH has both kinase and helicase activities
The previous binding of RNA pol II to TFIID occurred
through unphosphorylated C-terminal Domain (CTD)
of -subunit in the enzyme.
TFIIH (kinase) phosphorylates CTD to release RNA
pol II and activates the promoter clearance. Once the
polymerase has begun catalyzing phosphodiester
bond formation, then the complex is called an
initiation complex and allows a minimal level of
transcription to be initiated at a precisely defined site
downstream from the TATA box
Further enhancement (or repression) of that
transcription is achieved by interactions with
additional transcription factors that bind to upstream
promoter elements, enhancers, and silencers.
 Enhancers
a. can be located at great distances (>1000 bps)
from start site of transcription either from the 5'
or 3' end of gene
b. stimulates transcription (~100 times)
c. transcription factors bound to enhancer will
stimulate binding of RNAP II to promoter regions
closer to the start site of transcription.
 Elongation
It is carried out by RNA polymerase of the
initiation complex similar to the process of
prokaryotic transcription.
 Termination
The termination involves recognition of certain
sequence at the end of the gene without the
need for extra factors like prokaryotes.
END PART I
 Processing of mRNA precursor in
eukaryotes
Eukaryotic genes contain segments called introns,
which break up the amino acid coding sequence into
different segments called exons. In transcription, both
exons and introns are expressed to produce
preliminary large mRNA called heterogeneous RNA
(hnRNA ). The hnRNA contains sequences expressed
from both exons (coding regions) and introns (non
coding) regions on DNA.
This RNA must be processed in before it is translated
into the polypeptide.
 Processing of hnRNA

The processing of hnRNA occurs in the

nucleus and involves removal of the introns
sequences as well as the chemical
modifications of both RNA ends in order to
produce the mature RNA that will be functional in
translation.
 1. Removal of introns (RNA splicing)

Splicing is carried out by small nuclear

ribonucleoprotein complex(snRNPs )called the
spliceosome, which contains the following
components:
a. Special type of RNA called small nuclear RNA
b. Protein factors
c. Endonuclease
d. ligase enzyme.
The first step in RNA splicing is the formation of a
loop ,stabilized by the sequence present in small
nuclear RNA which is complementary to the two
splicing ends of an intron.
The endonuclease in the spliceosome complex will make cuts
at each end of the intron loop to release the intron while the
free ends of the two left exons are rejoined by the complex
ligase activity. The removed intron will be degraded, leaving
the exons sequence to be spliced together.
Splicing of mRNA precursors ( a lariat forms in the intron )

Fig. 11-34, p.295

 2. Capping of the 5 end in mRNA :( steps a-b)
Early in transcription when the growing RNA chain becomes
about 50 nucleotides long, an enzyme, guanyltransferase, adds
7-methylguanosine (m7Gppp) to the 5 end of the hnRNA. This
is called m7Gppp cap and the addition occurs in two steps.
First, a GTP is added to the 5'-end of RNA and the second
phase involves methylation of the guanine in the 7'-position to
generate the mature 7-methylguanosinetriphosphate cap
structure.
The cap structure is needed to protect the
mRNA from degradation by hydrolytic
enzymes and to help small ribosomal
subunits in recognizing the attachment
site on mRNA's 5' end.
3. Adition of Poly-A tail: :( steps c-f)
A recognition sequence of AAUAAA near the 3 end of
hnRNA helps to signal the removal of approximately 20
bp downstream of the recognition site by an
endonuclease enzyme. This is followed by addition of
poly(A) tail, made up of 150 to 200 repeating adenosine
residues, to the cleavage site at the 3 end. An enzyme
called poly(A) polymerase catalyzes this nucleotides
addition which results in the formation of complete
mature mRNA ready to leave the nucleus through
nuclear pores to the cytoplasm.The poly A tail increases
the stability of mRNA and prevents its degradation
during the export of mRNA to the ribosome.
The resulting mature mRNA may then exit the
nucleus and be translated in the cytoplasm.
 Alternative Splicing
It is the phenomenon of excluding or including certain
exons from the final RNA transcripts , the same Hn
RNA can be spliced in more than one mechanism to
produce different types of proteins with diverse
functions. Through this mechanism, it is possible for a
single gene to produce two or more different proteins.
In eukaryotes alternative splicing is considered a step
towards higher efficiency, because information can be
stored much more economically.
Alternative splicing occurs in more than 60% of the
human genes.
As a result, several types of protein with different
amino acid sequences can be synthesized
from such mRNA, each exhibiting different functions.
By exploiting this mechanism, one gene can produce
several protein types, thus functioning as if it were
several genes.
Control of transcription
via different subunit

Fig. 11-7, p.271

Elements of a bacterial promoter

Fig. 11-8, p.271

 Summary of eukaryotic transcription

It is more complicated than prokaryotic

transcription
3 RNA polymerases of which Pol II produces
mRNA
The organization of promoters & enhancers is
more complicated
An impoatant element is TATA box at -25
6 general initiation factors are involved in
forming the initiation complex
Four elements of polymerase II promoters in
eukaryotes

Fig. 11-17, p.280

DNA looping brings enhancers in contact with transcription factors and RNA
polymerase

Fig. 11-20, p.285

Table 11-4, p.286
Activation of transcription via CREB & CBP

Fig. 11-21, p.286

Multiple ways in which CREB binding protein (CBP) and p300 are involved in
gene expression

Fig. 11-22, p.287

CREB-The most important protein you never
heard of ?? Cell

Cell proliferation & differentiation

Mature T-lymphocytes
Metabolism
Pineal gland
Exercise
Memory & learning
Alzheimer
Table 11-5, p.287
Posttranscriptional modification of a tRNA precursor
Fig. 11-30, p.292
The organization of split genes in eukaryotes

Fig. 11-33, p.294

 Organization of the fast skeletal muscle troponin T gene
and the 64 possible mRNAs that can be generated from it

The exons in blue and red are exclusive .only one or the other may be used

Fig. 11-35, p.296

 SLE : Systemic lupus erythematosus is an auto immune disease
Production of a.b. to one of the snRNPs,U1-snRNP
Rash on the forehead & cheek bones, giving the wolf like appearance.
Severe kidney damage may follow with arthritis, accumulation of fluid
around the heart, & inflammation of the lungs.
SLE

p.296