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Md. Habibur Rahaman (HbR)
Dept. of Biochemistry and Microbiology
North South University
Central Dogma of Life
Transcription
The synthesis of RNA molecules using
DNA strands as the templates so that
the genetic information can be
transferred from DNA to RNA.
Similarity between
Replication and Transcription
• Both processes use DNA as the
template.
• Phosphodiester bonds are formed in
both cases.
• Both synthesis directions are from 5´
to 3´.
Differences between
Replication and Transcription
Replication
Transcription
template
double strands
single strand
substrate
dNTPs
NTPs
primer
yes
no
Enzyme
DNA polymerase
RNA polymerase
product
dsDNA
ssRNA
base pair
A-T, G-C
A-U, T-A, G-C
Differences between
Replication and Transcription
• During replication, all the nucleotides in the DNA template
are copied, but, during transcription, only small parts of the
DNA molecule—usually a single gene or, at most, a few
genes—are transcribed into RNA.
• Because not all gene products are needed at the same time
or in the same cell, it would be highly inefficient for a cell to
constantly transcribe all of its genes.
• Furthermore, much of the DNA does not code for a
functional product, and transcription of such sequences
would be pointless.
• Transcription is, in fact, a highly selective process—
individual genes are transcribed only as their products are
needed (Spatiotemporal: proper time and place)
Transcription: How is an RNA strand synthesized?
1.
Regulated by gene regulatory elements within each gene.
2.
DNA unwinds next to a gene.
3.
RNA is transcribed 5’ to 3‘
4.
Similar to DNA synthesis, except:

NTPs instead of dNTPs (no deoxy-)

No primer

No proofreading

Adds Uracil (U) instead of thymine (T)

RNA polymerase
X
Types of RNAs
Types of RNAs and Function
Different types of RNA, each encoded by different genes:
1. mRNA
Messenger RNA, encodes the amino acid sequence of
a polypeptide.
2. tRNA
Transfer RNA, transports amino acids to ribosomes
during translation.
3. rRNA
Ribosomal RNA, forms complexes called ribosomes
with protein, the structure on which mRNA is
translated.
4. snRNA
Small nuclear RNA, forms complexes with proteins
used in eukaryotic RNA processing (e.g., exon
splicing and intron removal).
5. miRNA/siRNA
Micro RNA/small interfering RNA, short ~22 nt RNA
sequences that bind to 3’ UTR target mRNAs and
result in gene silencing.
Template
• The whole genome of DNA needs to
be replicated, but only small portion
of genome is transcribed in response
to the development requirement,
physiological need and
environmental changes.
• DNA regions that can be transcribed
into RNA are called structural genes.
Template
•The template strand is the strand
from which the RNA is actually
transcribed.
•The coding strand is the strand
whose base sequence specifies the
amino acid sequence of the encoded
protein.
5'
GCAGTACATGTC
3' coding
3'
CGTCATGTACAG
5'
strand
template
strand
transcription
5'
GCAGUACAUGUC
3'
RNA
Asymmetric transcription
• Only the template fragment is used for
the transcription, but not the coding
fragment
• However, both strands can be used as
the templates.
• The transcription direction on different
strands is opposite.
• This feature is referred to as the
asymmetric transcription.
Asymmetric transcription
5'
3'
3'
5'
Asymmetric transcription
In most organisms, each gene is transcribed from
a single strand, but different genes may be
transcribed from different strands
Organization of coding information in
the adenovirus genome
A Gene is a Transcription Unit
A transcription unit includes a promoter, an RNA-coding region,
and a terminator.
Transcription in Prokaryotes
Only a single RNA polymerase
• In E.coli, RNA polymerase is 465 kD complex,
with 2 , 1 , 1 ', 1 
subunit
function

Determine the DNA to be
transcribed

Catalyze polymerization

Bind & open DNA template

Recognize the promoter
for synthesis initiation
E. coli RNA polymerase
Rifampicin, a therapeutic drug for tuberculosis
treatment, can bind specifically to the  subunit
of RNA-pol, and inhibit the RNA synthesis.
Transcription of Prokaryotes
• Initiation phase: RNA-pol recognizes
the promoter and starts the
transcription.
• Elongation phase: the RNA strand is
continuously growing.
• Termination phase: the RNA-pol stops
synthesis and the nascent RNA is
separated from the DNA template.
Prokaryotic promoter
3'
5'
-50
3'
-40
-30
-20
-35
region
TTGACA
AACTGT
-10
1
-10
region
TATAAT
ATATTA
(Pribnow box)
Consensus sequence
10
start
5'
Reaching A Consensus
Consensus sequences: Strongest promoters match consensus
Up mutation: mutation that makes promoter more like consensus
Down Mutation: virtually any mutation that alters a match with the consensus
Reaching A Consensus
Reaching A Consensus
These sequences (-10 and -35) are on the non-template
strand (coding strand/sense strand) and read 5’3’, left to
right.
Initiation
• RNA-pol recognizes the TTGACA
region, and slides to the TATAAT
region, then opens the DNA duplex.
• The unwound region is about 171 bp.
• The first nucleotide on RNA transcript
is always purine triphosphate. GTP is
more often than ATP.
• The pppGpN-OH structure remains on
the RNA transcript until the RNA
synthesis is completed.
• The three molecules form a
transcription initiation complex.
RNA-pol (2) - DNA - pppGpN- OH 3
• No primer is needed for RNA
synthesis.
• The  subunit falls off from the RNApol once the first 3-5 phosphodiester
bond is formed.
• The core enzyme moves along the
DNA template to enter the elongation
phase.
Prokaryotic Transcription Initiation
Sigma Recognizes the -10 and -35 Sequences
The structure of yeast RNA polymerase
reveals a groove that may be used by DNA as it
moves relative to the polymerase and a
potential groove for the newly formed RNA.
Mol Bio_Clark_Chapter: 06
Promoter Strength
The three important regions :
(i) the 35 consensus region
(ii) the 10 consensus Pribnow sequence
(iii) the initiation site.
Nucleotide composition (AT-rich regions separate
more easily than GC-rich regions)
Degree of supercoiling
Interference of topoisomerases
The conformation of the DNA
Facts to know
•The start site itself is not marked by a consensus
sequence but often has the sequence CAT
•The position of the start site is determined not by the
sequences located there but by the location of the
consensus sequences
•If the consensus sequences are artificially moved
upstream or downstream, the location of the starting
point of transcription correspondingly changes.
Elongation
• The release of the  subunit causes
the conformational change of the
core enzyme. The core enzyme slides
on the DNA template toward the 3
end
• Free NTPs are added sequentially to
the 3 -OH of the nascent RNA strand.
(NMP)n + NTP
RNA strand
substrate
(NMP)n+1 + PPi
elongated
RNA strand
• RNA-pol, DNA segment of ~18nt and
the nascent RNA form a complex
called the transcription bubble
• The 3 segment of the nascent RNA
hybridizes with the DNA template, and
its 5 end extends out the
transcription bubble as the synthesis
is processing
• This rate of RNA synthesis is much
lower than that of DNA synthesis
Transcription bubble
About 8 nucleotides of newly synthesized RNA are paired with the DNA-template
nucleotides at any one time
RNA-pol of E. Coli
RNA-pol of E. Coli
As the transcription apparatus moves down the DNA template, it generates positive supercoiling
ahead of the transcription bubble and negative supercoiling behind it
Termination
(i) RNA polymerase must stop synthesizing RNA
(ii)The RNA molecule must be released from RNA polymerase
(iii)The newly made RNA molecule must dissociate fully from the DNA
(iv)RNA polymerase must detach from the DNA template
Transcription ends after the terminator has been
transcribed, like a car that stops only after running over a
speed bump.
The termination occurs in either  -dependent or  independent manner
-independent termination
•
The termination signal is a stretch
of 30-40 nucleotides on the RNA
transcript, consisting of many GC
followed by a series of U.
•
The sequence specificity of this
nascent RNA transcript will form
particular stem-loop structures to
terminate the transcription.
•
The adenine–uracil base pairings
downstream of the hairpin are
relatively unstable and the
formation of the hairpin may itself
destablize the DNA–RNA pairing
-dependent termination
The  factor, a hexamer, is a
ATPase and a helicase
Summary
•
•
•
•
•
•
•
•
Transcription is a selective process
RNA is transcribed from single-stranded DNA
NTPS are used as the substrates in RNA synthesis
RNA molecules are antiparallel and complementary to
the DNA template strand
Transcription depends on RNA polymerase—a
multimeric enzyme
The core enzyme requires a sigma factor in order to bind
to a promoter and initiate transcription
Promoters contain short sequences crucial in the binding
of RNA polymerase
Transcription ends after a terminator has been
transcribed
Transcription of Eukaryotes
• Differ from prokaryotes
(i) Eukaryotic cells possess three different RNA
polymerases, each of which transcribes a
different class of RNA and recognizes a different
type of promoter
(ii) Another difference is in the nature of promoter
recognition and initiation. Many proteins take
part in the binding of eukaryotic RNA
polymerases to DNA templates, and the
different types of promoters require different
proteins.
Transcription of Eukaryotes
Transcription of Eukaryotes
• Need to gain access to DNA-Histone complex
(i) Acetyltransferases add acetyl groups to amino
acids at the ends of the histone proteins, which
destabilizes the nucleosome structure and
makes the DNA more accessible
(ii) Proteins called chromatin-remodeling proteins
may bind to the chromatin and displace
nucleosomes from promoters and other regions
important for transcription
Transcription of Eukaryotes
• Transcription initiation needs
promoter and upstream regulatory
regions.
• The cis-acting elements are the
specific sequences on the DNA
template that regulate the
transcription of one or more genes.
Cis-acting element
cis-acting element
structural gene
GCGC
CAAT
TATA
exon
intron exon
start
TATA box
enhancer
GC box
CAAT box
(Hogness box)
Transcription factors
• RNA-pol does not bind the promoter
directly.
• RNA-pol II associates with six
transcription factors, TFII A - TFII H.
• The trans-acting factors are the
proteins that recognize and bind
directly or indirectly cis-acting
elements and regulate its activity.
Transcription factors
• General transcription factors, which, along
with RNA polymerase, form the basal
transcription apparatus that assembles
near the start site and is sufficient to
initiate minimal levels of transcription.
• Transcriptional activator proteins, which
bind to specific DNA sequences and bring
about higher levels of transcription.
Promoters
• Core Promoter: located immediately upstream of the gene and has
one or more consensus sequences.
1.
2.
3.
4.
The most common of these consensus sequences is the TATA
box, which has the consensus sequence TATAAA and is located
from 25 to 30 bp upstream of the start site.
TFIIB recognition element (BRE), which has the consensus
sequence G/C G/C G/C C G C C and is located from 32 to 38
bp upstream of the start site.
Instead of a TATA box, some core promoters have an initiator
element (Inr) that directly overlaps the start site and has the
consensus Y Y A N T/A Y Y.
Downstream core promoter element (DPE) is found
approximately 30 bp downstream of the start site in many
promoters that also have Inr; the consensus sequence of DPE
is R G A/T C G T G.
Promoters
Core Promoter
Core promoter are recognized by transcription factors
that bind to them and serve as a platform for the
assembly of the basal transcription apparatus
Promoters
• Regulatory Promoter:
• Transcriptional activator proteins bind to these sequences,
affect the rate at which transcription is initiated
• Some regulatory promoters also contain repressing
sequences, which are bound by proteins that lower the rate
of transcription
Assembly of basal transcription apparatus
Assembly of basal transcription apparatus
• TFIID binds to the TATA box and positions the active
site of RNA polymerase II
• TFIID consists of at least nine polypeptides. One of
them is the TATA-binding protein (TBP)
• TFIIA helps to stabilize the interaction between TBP
and DNA
• TFIIB plays a role in the selection of the start site
• TFIIH has helicase activity and unwinds the DNA
during transcription
• The mediator plays a role in communication
between the basal transcription apparatus and
transcriptional activator proteins
Enhancers
• DNA sequences that increase the rate of transcription
at distant genes are called enhancers
• Enhancers can stimulate any promoter in their
vicinities
• Enhancer may be upstream or downstream from the
affected gene or, in some cases, within an intron of the
gene itself
• Enhancers also contain sequences that are recognized
by transcriptional activator proteins
• Enhancers like sequences sometimes take part in
repressing transcription called silencers.
Termination
• RNA polymerase I requires a termination factor, like the rho
factor utilized in termination of some bacterial genes. Unlike
rho, which binds to the newly transcribed RNA molecule, the
termination factor for RNA polymerase I binds to a DNA
sequence downstream of the termination site.
• RNA polymerase III ends transcription after transcribing a
terminator sequence that produces a string of Us in the RNA
molecule, like that produced by the rho-independent
terminators of bacteria. Unlike rho-independent terminators
in bacterial cells, RNA polymerase III does not require that a
hairpin structure precede the string of Us.
• In many of the genes transcribed by RNA polymerase II,
transcription can end at multiple sites located within a span of
hundreds or thousands of base pairs.
Thank you!