RNA

transcription

Lec 6th
Molecular biology
According to central dogma , the genetic information flow from DNA the RNA
via transcription process then it will translate later to functional protein.
Transcription is a process of making an RNA strand from a DNA
template, and the RNA molecule that is made is called transcript.
Transcription like replication need free 3́ end to add the complementary
nucleotide ,also the direction of movement and polymerization of RNA
polymerase from 5́à3́ .
 Iden%fica%ons :
1- promoter: regulatory nucleotide sequences of DNA (40-60 nts) at the
beginning of every gene locate upstream (towards the 5' region) of a gene
on the template or sense strand. In this site, the two DNA strands will be
opened but not translated. The promoter provides a control point for
regulated gene transcription.
In prokaryotes, the promoter is recognized by RNA polymerase
(δ sigma subunit) because it has a great affinity to bind to this region. There
are great differences between the promoter area of Prokaryotic and
Eukaryotic cell as it is divided into many sub-regions.
2- Coding region: Nucleotide sequences which will detriment the genetic code
then will be translated to amino acid. It starts with ATG triplet initiation codon
(AUG in mRNA). The length of coding region depend on the type of
produced protein
3- Terminator: Nucleotide sequences exist after the coding region rich with poly
G followed by poly C then poly A (in Prokaryotes), while in Eukaryotic cells,
Termination begins when a polyadenylation signal appears in the RNA
transcript. This is a sequence of nucleotides that marks where an RNA
transcript should end. The polyadenylation signal is recognized by an
enzyme that cuts the RNA transcript nearby, releasing it from RNA
polymerase.
Difference between Eukaryotic and Prokaryotic Promoters
1- Prokaryotic promoter
The promoter consists of two short sequences known as -10 box and
-35 box positions upstream from the transcription start site.
• The sequence at -10 box is also called the Pribnow box (according to
David Pribnow who discover this elements 1975) , or the -10 element,
and usually consists of the six nucleotides TATAAT. The Pribnow box
is absolutely essential to start transcription in prokaryotes since it rich
with T & A nitrogenous bases .
• The other sequence is -35 box (-35 element) that consists of the six
nucleotides TTGACA. RNA polymerase attached here via its sigma
subunit. The two boxes separated by 17 ±1 base pairs .
There are differences between the promoter region in pro and Eukaryotic cells
and for sure this promoter region will not translate to protein.
Eukaryotic promoters more complicated and divided into many sub regions
1- regulatory promoter to regulate promoter activity.

2-core promoter: divided into many region as with prokaryotic promoter

A- recognition element at -35 box (in other reference -30 or -75 )in all cases it
is rich with GC nts thus DNA will not be opened here.
B- TATA box at -25 box (in other references at -26 or -29 ) as with -10 box in
prokaryotic it is rich with AT nts thus the two DNA strand will be opened here
(it located 25 nts far away from the start point +1). Discovered by David
Hogness in 1977 thus it is called Hogness box. RNA polymerase will bind to
-35 box but the two strand is opened here.
C-initiator elements (Inr) : represent transcription start point (from -2 to +4)
including +1
D-Downstream core promoter element (DPE): at +20 to +32 region
Transcription steps :As with replica%on ,the 3 steps are:
1-Ini%a%on 2- Elonga%on 3- Termina%on
The RNA polymerase (RNAP) enzyme is composed of a core and a
holoenzyme structure. The core enzyme consist from ββ’α2ω
subunits.
The holoenzyme is composed of core enzyme and a specific
component known as sigma δ70 which is more predominant type in E.
coli.
When all the 6th subunits are present, RNA polymerase is in its
active form and is referred to as the holoenzyme, but when the σ-
factor detaches, it is in core polymerase form. Each subunit plays a
role in the initiation of transcription, and the σ-factor must be present
for initiation to occur. The sigma factor functions in aiding in promoter
recognition, correct placement of RNA polymerase, and beginning
unwinding at the start site.
 First step: Initiation stage
• In bacteria, binding of RNAP involves recognizing promoter region
by sigma factor then binding to -35 box forming closed complex. The
α subunit C-terminal domain (αCTD) helps in binding to promoter
upstream elements. Usually in E. coli, σ70 is expressed under normal
conditions and recognizes promoters for genes required under normal
conditions (house-keeping genes), while σ32 recognizes promoters for
genes required at high temperatures (heat-shock genes).

• After binding to the DNA, the RNAP switches from a closed complex to
an open complex at -10 box. This change involves the separation of
the DNA strands to form an unwound DNA strand of approximately 13
bp, referred to as the transcription bubble. Only one strand of DNA,
called the template strand (also called the noncoding strand or
nonsense/antisense strand), gets transcribed. Ribonucleotides are
base-paired to the template DNA according to Watson-Crick base-
pairing interactions.

• Super coiling plays an important part in polymerase activity because of

the unwinding and rewinding of DNA. Usually regions of DNA in front of
RNAP are unwound while regions behind RNAP are rewound and
negative super coiled are present.
 RNAP

DNA

Schematic representation of bacterial RNAP holoenzyme, σ factor structure, and

representative promoter structure.
-35 -10

Coding strand

Template strand
 Second step: Elongation
• Transcription elongation involves the further addition of ribonucleotides and
change of the open complex (at -10 box) to transcriptional complex. RNAP
cannot start forming full length RNA transcript because of its strong binding
to the promoter. Therefore, it must leave promoter region and further
progress. Transcription at this stage primarily results in short RNA
fragments of around 10-9 bp. Once the RNAP starts forming longer
transcript after leaving the promoter. The σ factor falls off RNAP that allows
the core RNA polymerase to move forward.

• Ribonucleosides are attached to the OH- molecule on the 3' end of the
RNA, transcription always occurs in the 5’à3' direction. Multiple RNA
polymerases can be active at once, meaning many strands of mRNA can
be produced very quickly. RNAP moves down the DNA rapidly at
approximately 40 bases per second.

• Although RNAP does not seem to have the 3’ exonuclease activity that
characterizes the proofreading activity found in DNA polymerase, there is
evidence of that RNAP will halts at mismatched base-pairs and correct it.
This figure shows transcriptional bubbles moving along the DNA template and the
nascent RNA gets longer, growing from 5à3 direction. Notice that all T nitrogen
base will replace by U in the transcribed RNA
 Third step : Termination
In prokaryotes can be rho-independent or rho-dependent
1-Rho-independent transcription termination:
Intrinsic termination (also called Rho-independent termination) is a
mechanism in prokaryotes that causes RNA transcription to be stopped
without the aid of rho protein.
This process is controlled by specific sequences known as terminator or
attenuator in the DNA template strand. As the polymerase reaches the end
of the gene being transcribed, it encounters a region rich in C–G
nucleotides followed by the poly-A region.
The mRNA folds back on itself, and the complementary C–G nucleotides
bind together, making it more stable than the DNA-RNA hybrid itself.
The result is a stable “hairpin” structure that causes the polymerase to stall
as soon as it begins to transcribe a region rich in A–T nucleotides called
stem-loop. The complementary U–A region of the mRNA transcript forms
only a weak interaction with the template DNA leading to lower the energy
of destabilization for the RNA-DNA duplex, allowing it to unwind and
dissociate from the core enzyme to break away and liberate the new mRNA
transcript.
Intrinsic Termination: An inverted repeat sequence near the end of
the newly synthesized strand of RNA causes the formation of a hairpin
loop.
2- Rho-dependent transcription termination (Rho factor):
is a prokaryotic ATP-dependent unwinding enzyme involved
in termination transcription.

In this mechanism, Rho protein is necessary to induce termination. Rho

protein activity depends on the Rho Utilizing sequence (rut sequences);
~40 nucleotides long rich with C) and uses the energy obtained from the
ATP hydrolysis to translocate along the RNA.

Bacterial Rho protein is a hexameric (ring-shaped protein) RNA-DNA

helicase that serves as a general bacterial transcription termination. It
binds to ssRNA and uses its ATPase activity to induce termination.

Rho binds to RNA and then uses its ATPase activity to provide the
energy to translocate along the RNA until it reaches the RNA–DNA
helical region, where it unwinds the hybrid duplex structure.
(rut sequences)
 General differences between transcrip%on and replica%on:
[Link] [Link]

purpose The purpose of replica%on is to conserve The purpose of transcrip%on is to

the en%re genome for next genera%on. make RNA copies of individual genes
that the cell can use in the metabolic
ac%vity
Defi[Link] DNA replica%on is the replica%on of a synthesis of RNA from a DNA
strand of DNA into two daughter strands, t e m p l a t e . 3 t y p e s w i l l
each daughter strand contains half of the transcribed ,rRNA and tRNA are final
original DNA double helix. product ,mRNA will translated to
protein
products One strand of DNA becomes two daughter mRNA, tRNA, rRNA and non-coding
strands. RNA( like microRNA)
Timing It happened once the cell start division At any %me in the cell as required
Enzyme and The two strands are separated and then the codons of a gene are copied into
results each strand's complementary DNA messenger RNA by RNA polymerase.
sequence is synthesized by DNA By the help of tRNA, which carries
polymerase. The process required primers. amino acids and rRNA the codon will
translated to protein. The process
required promoter
differences [Link] [Link]
Ini%a%on primer ,DnaA box & ini%a%on protein Require promoter region
need
Template The 2 strands serve as a template Only template strand(3→5)
Involved area The total genome will replicate precise part will transcribed