Transcription processes

1. TRANSCRIPTION
RNA polymerase carries out transcription. Transcription is the process of linking ribonucleotide to
form long chain or to form oligonucleotide. The process of synthesizing
RNA from DNA molecule.
The 2’-OH of the ribose sugar make phosphodiester bond unstable and
that causes ssRNA easy to be degraded. Cell has mechanism to remove
mRNA that is not correct.
Transcription VS Replication
-
Transcription initiates both of prokaryotic and eukaryotic cell
Form more sites than replication
The number of RNAP molecules are more than number of
DNAP molecules
RNAP proceed at slower rate than DNAP (~ 50-100 bases/sec for RNAP, ~ 1000 bases/sec for
DNAP)
The fidelity of RNAP lower than DNAP
 Allowed since the aberrant RNA molecules can simply be turned over and new correct
molecules can be made. (true for Prok. But not Euk.)
**Replication happens too fast and cause lots of error but DNAP has exonuclease activity to correct
the error.
Transcription has three steps (Initiation, Elongation and Termination)
Before proceeds to the steps, let us look at the Promoter.
Promoter is a region of DNA that initiates transcription of a particular gene. Promoters are located
near the genes they transcribe, on the same strand and upstream on the DNA (towards the 3' region
of the anti-sense strand, also called template strand and non-coding strand). Promoters can be about
100–1000 base pairs long.
Promoters contain specific DNA sequences and response elements that provide a secure initial
binding site for RNA polymerase and for proteins called transcription factors that recruit RNA
polymerase.
In bacteria - The promoter is recognized by RNA polymerase and an associated sigma factor, which in
turn are often brought to the promoter DNA by an activator protein's binding to its own DNA binding
site nearby.
In eukaryotes - The process is more complicated, and at least seven different factors are necessary
for the binding of an RNA polymerase II to the promoter.
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2. TRANSCRIPTION
In bacteria, the promoter contains two short sequence elements approximately -10 and -35
nucleotides upstream from the transcription start site.


The sequence at -10 (the -10 element) has the consensus sequence TATAAT.
The sequence at -35 (the -35 element) has the consensus sequence TTGACA.
The nucleotide at which transcription is initiated is denoted as +1. The preceding nucleotide as -1.
Those portions of the DNA preceding the initiation site (toward the 3’ end of the template) are said
to be upstreamfrom that site. Those portions of the DNA succeeding it (toward the 5’ end of the
template) are said to be downstream from that site.
The RNAP is a holoenzyme that consist of 5 subunits
Rifampicin – a semi synthetic antibiotic derived from the original molecule (rifamycin – isolated from
streptomycete). Binds to the beta (β) subunit of RNAP and inhibits the formation of first
phosphodiester bond. Act on prok. and euk.
The sigma (σ) factor
-
Proteins that are required for the binding of the RNA polymerase to the promoter so the
transcription can be initiated.
Recognizes promoter and facilitates initiation.
Facilitate the binding of RNAP to dsDNA.
To direct RNAP to transcribe DNA template at the correct transcription start site (TSS).
Function of sigma (σ) factor
-
Ensure RNAP binds stably to DNA only at promoter (not string bind) at first but then if reach
prmoter sequence the strong binding)
Destabilize nonspecific binding to nonpromoter DNA
Stabilize specific binding to promoter DNA
Accelerates the search for promoter DNA
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3. TRANSCRIPTION
RNA polymerases are not capable of recognizing promoters on their own but require the help of
additional proteins called transcription factors
(in Eukaryotes)
The polymerase moves along the template DNA
strand towards its 5’ end (3’  5’ direction).
Therefore, the resultant pre-mRNA transcript
grows from the 5’ end. The DNA is temporarily
unwound as the polymerase progresses and
reform the double helix after the polymerase
has moved past a particular stretch of DNA.
Chain elongation occurs as a result of an attack by
3’ OH of the nucleotide at the end of the growing
strand on the 5’ α-phosphate of the incoming
nucleoside triphospahte
For each gene, only one of the two DNA strand is
transcribed. This strand is called the template
strand. Note that for every particular gene, the
same strand is used as the template every time it
is transcribed. The mRNA base triplets are called
codons. the nontemplate strand is sometimes
called the coding strand. The nontemplate
sequences are same as the sequence of mRNA
except for the T is replaced by U.
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4. TRANSCRIPTION
Transcription in Prokaryotes
As what has been mentioned before, bacteria such
as E.coli contain RNA polymerase composed of 5
subunits. Without the σ subunit, it is called as core
enzyme. Before the enzyme (RNAP) attaches itself to
the DNA, σ factor is added. Attachment of σ factor
to the core enzyme increases the enzyme’s affinity
for promoter sites in DNA and decreases its affinity
for DNA in general. Once bound to the promoter,
the enzyme separates (melt) the two DNA strands in
the region surrounding the start site, which make
the template strand accessible to the enzyme’s
active site. One approximately ten nucleotides have
been successfully incorporated into a growing
transcript; the enzyme undergoes a conformation
change which lead to the release of σ sigma factor
and the promoter DNA. This will make the enzyme
more stable and is converted into an elongation
complex.
Bacterial cells possess a variety of different σ factors that
Recognize different versions of the promoter sequence. The σ70 is known as the “housekeeping” σ
factor, because it initiates transcription of most genes. Alternative σ factors initiate transcription of a
small number of specific genes that participate in a common response. For example, when E. coli
cells are subjected to a sudden rise in temperature, a new σ factor is synthesized that recognizes a
different promoter sequence and leads to the coordinated transcription of a battery of heat-shock
genes.
To terminate the transcription, a specific nucleotide sequence is needed. Terminations involve two
cases, which are rho-dependent termination and rho-independent termination (also known as
intrinsic termination).
Rho (ρ)-independent termination
These termination sequences have two characteristics
features:
-
A series of U residues in the transcribed RNA
GC rich self-complementary region with several
intervening nucleotides
When the self-complementary region of a growing RNA
chain is synthesized, the complementary sequences
base-pair with one another, forming a stem-loop structure (hairpin loop).
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5. TRANSCRIPTION
This stem-loop structure is thought to interact with the transcribing E.coli RNA polymerase, causing it
to pause. The base pair between the U residues at the 3’ end of the nascent RNA chain and the A
residues in the template DNA strand are extremely unstable compared to other types of WatsonCrick base pairs. If the base pairs is replaced or substituted with other bases, the terminator function
may decreases because the RNA-DNA hybrid duplex will be more stable. These two features probably
permit release of the RNA chain from the transcription complex at Rho-independent termination
sites.
Rho (ρ)-dependent termination
-
-
Rho factor is a hexameric protein which wraps around the 70-to 80-base segment of the
growing RNA transcript
Activates the ATPase activity of Rho that is associated with its movement along the RNA in
the 3’ direction until it eventually unwinds the RNA-DNA hybrid (break H-bond) at the active
site of RNAP.
Pausing of polymerase during elongation is thought to be an important component of Rhodependent termination as it is in Rho-independent termination.
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