Lecture.6 DNA Transcription

MOLECULAR BIOLOGY & GENETICS
Basic Processes of Molecular Biology
DNA Transcription
6th Lecture
Qurat-ul-Ain, B. Pharm., M. Phil., Ph.D.,
Visiting Assistant Professor
Department of Physical Therapy, Faculty of Health& Medical Sciences,
Hamdard University, Karachi, Pakistan
Assistant Professor
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Hamdard
University, Karachi, Pakistan.
Qurat-ul-Ain, B. Pharm., M. Phil., Ph.D., qurat.fophu@yahoo.com

RNA Transcription
• RNA polymerase binds to the bacterial DNA in spe region known as promoter
• The polymerase, using its σ factor recognizes this DNA seq by making specific
contacts with the portions of the bases that are exposed on the outside of the helix
• After RNA polymerase binds tightly to the promoter DNA in this way, it opens up
the double he;lix to expose short stretches of nt on each strand
• Instead of DNA helicase, here the nick does not require the hydrolysis of ATP,
both DNA and polymerase structurally changes themselves in a more energetically
favor state.
• One DNA act as a template for the incoming ribonucleotides, joined by RNA
polymerase
• After few additions of nt on newly RNA, polymerase works without its sigma
factor and goes in to more structurally stable state.
•50 nt/ sec is added by bacterial RNA polymerase, and it continues until it
recognized the terminator seq

• RNA polymerase I transcribed 5.8 S, 18S, and 28S rRNA genes
• RNA polymerase II transcribed all protein-coding genes,plus snoRNA
genes and some snRNA genes
• RNA polymerase III transcribed tRNA genes, 5S rRNA genes, some
snRNA genes and genes for other small RNAs
RNA polymerases in Eukaryotic cells

In Eukaryotes: RNA polymerase II
• RNA polymerase II along another
transcription factors are required for the
correct position at the promoter side on
the DNA strand.
• The transcription factors helps in pulling
apart the DNA strand to allow
transcription to begin and release RNA
polymerase from the promoter into
elongation mode
• The general transcription factors are
called as TFII, TFIIA, TFIIB (transcription
factor for polymerase II)

Initiation of the RNA transcription by RNA polymerase II
• All the transcription factors assembled on the
promoter region which is recognized by the RNA
ployII
• First TFIID binds to short stretch of DNA helix,
composed of T and A or TATA box and to TATA
binding protein (TBP)
• TATA box is localized 25nt upstream from the
transcription start
• It is not the only seq at which initiation starts but
RNA polymerase II prefers this seq much better
• For transcription initiation complex, TFIID
creates a big loop in the DNA strand so that all
the factors can join and assembled at the
promoter region for the protein assembly steps.

• TFIIH has a kinase activity as well as a helicase
property
• The termination of RNA is carried out TFIIH
which adds phosphate groups to the tail of the
RNA polymerase known as CTD or C-terminal
domain
• RNA polymerase then disengage form the
initiation complex
• Undergo many structural conformations and
binds tightly with the DNA , binds to many other
proteins and start the synthesis of RNA for a
longer distances
• PO4rlylation important in that sense that it
signals to the RNA processing units as it comes
from the polymerase

RNA processing (Post transcriptional modification)
The basic features of transcription are the same in
prokaryotic and eukaryotic cells, but eukaryotic genes and
their mRNA molecules are more complex than those of
bacteria. Eukaryotic mRNA molecules undergo
posttranscriptional modifications, which apparently protect
eukaryotic mRNA molecules from degradation and to give
them longer lifetimes than bacterial mRNA.
A DNA sequence containing both exons (coding regions)
and introns (noncoding regions) is transcribed by RNA
polymerase to make the primary transcript, or mRNA
precursor. As it is synthesized, the pre-mRNA is capped
by the addition of a modified base to its 5' end (5'
capping) and a poly-A tail (100 to 250 nucleotides long)
is added to the 3' end. Introns are later removed from the
mRNA precursor. Exons are spliced together to produce a
continuous protein-coding sequence. Finally, mature
mRNA is transported through the nuclear envelope and
into the cytosol to be translated.

The pre-mRNA molecule undergoes three main
modifications. These modifications are 5' capping, 3'
polyadenylation, and RNA splicing, which occur in the cell
nucleus before the RNA is translated.
5' Processing
Capping of the pre-mRNA involves the addition of 7-
methylguanosine (m7G) to the 5' end. To achieve this, the
terminal 5' phosphate requires removal, which is done with
the aid of a phosphatase enzyme. The enzyme guanosyl
transferase then catalyses the reaction, which produces the
diphosphate 5' end. The diphosphate 5' prime end then
attacks the α phosphorus atom of a GTP molecule in order to
add the guanine residue in a 5'5' triphosphate link. The
enzyme S-adenosyl methionine then methylates the guanine
ring at the N-7 position. This type of cap, with just the (m7G)
in position is called a cap 0 structure. The ribose of the
adjacent nucleotide may also be methylated to give a cap 1.
Methylation of nucleotides downstream of the RNA molecule
produce cap 2, cap 3 structures and so on. In these cases
the methyl groups are added to the 2' OH groups of the
ribose sugar. The cap protects the 5' end of the primary RNA
transcript from attack by ribonucleases that have specificity
to the 3'5' phosphodiester bonds.

3' Processing
Cleavage and Polyadenylation
The pre-mRNA processing at the 3' end of the RNA molecule involves
cleavage of its 3' end and then the addition of about 200 adenine residues to
form a poly(A) tail. The cleavage and adenylation reactions occur if a
polyadenylation signal sequence (5'- AAUAAA-3') is located near the 3' end of
the pre-mRNA molecule, which is followed by another sequence, which is
usually (5'-CA-3'). The second signal is the site of cleavage. A GU-rich
sequence is also usually present further downstream on the pre-mRNA
molecule. After the synthesis of the sequence elements, two multisubunit
proteins called cleavage and polyadenylation specificity factor (CPSF) and
cleavage stimulation factor (CStF) are transferred from RNA Polymerase II to
the RNA molecule. The two factors bind to the sequence elements. A protein
complex forms that contains additional cleavage factors and the enzyme
Polyadenylate Polymerase (PAP). This complex cleaves the RNA between the
polyadenylation sequence and the GU-rich sequence at the cleavage site
marked by the (5'-CA-3') sequences. Poly(A) polymerase then adds about 200
adenine units to the new 3' end of the RNA molecule using ATP as a precursor.
As the poly(A) tails is synthesised, it binds multiple copies of poly(A) binding
protein, which protects the 3'end from ribonuclease digestion.[3]

Splicing
RNA splicing is the process by which introns, regions of RNA that do
not code for protein, are removed from the pre-mRNA and the
remaining exons connected to re-form a single continuous molecule.
Although most RNA splicing occurs after the complete synthesis and
end-capping of the pre-mRNA, transcripts with many exons can be
spliced co-transcriptionally.[4] The splicing reaction is catalyzed by a
large protein complex called the spliceosome assembled from proteins
and small nuclear RNA molecules that recognize splice sites in the pre-
mRNA sequence. Many pre-mRNAs, including those encoding
antibodies, can be spliced in multiple ways to produce different mature
mRNAs that encode different protein sequences. This process is
known as alternative splicing, and allows production of a large variety
of proteins from a limited amount of DNA.

RNA Capping
• The 5’ end of the new RNA molecule is modified by the addition of a
cap contains a modified Guanine nucleotide
• The capping reaction is catalyzed by
1- Phosphatase which removes a PO4 from 5’ end of the RNA
2- a guanyl transferase that adds GMP in a reversal linkage 5’-5’
instead to 5’-3’
3- Methyl transferase that adds a methyl group to the guanosine
• These enzyme binds the PO4rylated RNA as soon as it synthesized
from the polymerase and will modify 5’ end of the RNA
• The 5’ methyl cap signals the 5’ end of eukaryotic mRNAs- this is to
distinguish the RNA from the other types of RNA mols in the cells
• For eg RNA polymerase I and II produce uncapped RNAs during
transcription, because they lack tails
• In nucleus the cap binds a protein complex called CBC (cap-binding
complex), that helps RNA to processed and export out in the cytosol
properly
• 5’ methyl cap is very important in translation of mRNAs in the
cytosol

mRNA molecule

5’ Capping of the mRNA

RNA factory

Removal of Introns from the newly synthesized Pre- mRNA
• Eukaryotes genes were found in many
coding sequence known as expressed or
axon and intervening sequences or introns
• However both are transcribed in to RNA
• During RNA Splicing these introns are
removed from the RNA and only axons are
joined back
• After 5’ and 3’ end have been processed,
and only those mols. of Pre-RNA goes in to
splicing who r destined to become mRNA
• One splicing removes only one intron
• In this two sequential phosphoryltransfer
reaction or transesterfications join the exons
while removing the introns as a “lariat”

RNA Splicing Maschinery
• It consists of 5 additional RNA
mols. + > 50 proteins + requires
many ATP mols/ splicing
• This event should be highly
accurate, any mistake in splicing
could harm or kill the cells
• Importance of introns= To
produce new types of proteins +
helps in genetic recombination to
combine the axons of different
genes + with the same genes
• emergence of Protein domains
• Due to mRNA splicing many
different types of mRNA and
proteins are produce
• RNA splicing also helps cells to
change the nature of its gene
expression

• Introns can be 10 nt to 100,000 nt long
• Picking the correct place for their removal
is not easy
• This is done at 3 positions
1- 5’ splice site
2- 3’ splice site
3- Branch point at the middle of the intron
(excised lariat)
• These all positions have similar
consensus nt sequences which mark
splicing positions
• Still difficult to remove all
Splicing positions on Introns

Spliceosome
• RNA molecules are involved instead of
proteins for splicing
• Short RNA mols (200 nt) and named U1,
U2, U4, U5, U6
• They all form a complex called as snRNAs
(small nuclear RNAs)
• snRNAs work with 7 protein subunits,
snRNP (small nuclear ribonulceo protein)
• snRNAs + snRNPs forms the core of
spliceosome
• The recognition b/w 5’ and 3’ splice
junction, branch point take place b/w Pre-
mRNA substrate and consensus RNA nt
sequences
• RNA-RNA rearrangement occur in which
U1 is replaced by U6 at 5’ and so on
• This permits the checking and rechecking
of RNA seq before the chemical reaction is
allowed to proceed.
• This enhance accuracy

mRNA splicing mechanism

Complex RNA-RNA Rearrangements
• ATP is required for the assembly of the spliceosome
• RNA helicase are used to break up the RNA-RNA
interaction and need ATP
• Total 50 proteins for each splicing event
• ATP-requiring RNA-RNA rearrangements occur within
themselves and b/w the snRNPs and the Pre-mRNA
• Active site on Pre-mRNA is created after the
rearrangements
• Catalytic site:
1- It is made of RNA mols
2- U1 and U2 forms a 3 dimensional structure in
spliceosome that juxtaposes the 5’ end splice site of Pre-
mRNA
3- This site is also attched with the branch point
4- Causes the 1st Esterfication reaction
5- This brings 5’ and 3’ splice junctions close and requires
U5 snRNA for the 2nd esterfication
6- At the end the product is released but snRNPs remian
attached. Same procedure starts again

RNA-RNA rearrangements

Proper splice sites in Pre-mRNA
• Soon after the transcription and the 5’ cap formation
several functions of spliceosome acts on the
PO4rylated tail of the RNA polymerase
1- Pre-mRNA coming from RNA polymerase keeps
the track of intron and exons
• The 5’ snRNP with only one 3’splice site
•This helps to prevent wrong exon skipping
2- Exon definition hypothesis
• RNA synthesis proceeds with SR proteins act like a
component of spliceosome
• They mark 3’ and 5’ splice site starting from 5’ end
of RNA
• This involves U1 snRNA, mark the exon boundary
and U2AF, help to specify other
• These complexes on nascent RNA prevents cryptic
sites
• Splicing is done while RNA is still synthesizing from
RNA polymerase, this means that splicing can be
done along transcription or after the transcription

snRNPs splice a small fraction of Intron sequences
• A small set of snRNPs that direct spliceosome
recognizes only specific set of DNA sequences, AT-AC
spliceosome
• However same RNA-RNA rearrangements are present
Another variation of splicing mechanism exits called as
trans-splicing
• In this exons from two separate RNA transcripts are
spliced together to form a mature mRNAmols.
• Trypanosomes produce all their mRNA in this way,
however few nematode mRNA are produce by
transplicing
1- one exon is spliced in to 5’ end of many different RNA
transcripts produce by the cell
2- This way all RNA have same 5’ but diff 3’ ends
3- trans-splicing uses snRNP, SLRNP
• Importance is that may be same 5’ site enhances the
translation of mRNA

RNA splicing and Plasticity
• When a mutation occurs in a nucleotide seq critical for
splicing of a particular intron, it did not splice that intron
• The exon will be skipped
• New pattern of splicing comes inaction and creates a
cryptic junctions and picks out the best pattern of splice
junctions
• If the present one got mutated it will seek out a new
one having best pattern
• This flexibility in the process of RNA splicing suggest
that mutations and changes in pattern are important in
evolution of genes and organisms
• Plasticity also refers that the cell can easily regulate
the pattern of RNA splicing
• Alternative splicing give rise to many different types of
proteins belong to the same gene. They are
constitutive in the cells.
• Some times their expressions are regulated by the
cells

RNA-Processing Enzymes Generate the 3’ end of Eucaryotic mRNAs
• RNA polymerase continues its movement along gene,
the spliceosome on the RNA and cut the intron and
axon boundaries
• The long C-terminal tail of the RNA polymerase make
sure that all the components for the splicing should be
present on the RNA
• It also initiates the 3’ end of the mRNA for the
processing
• Processing starts after getting signals from DNA to
transcribe in to RNA as the RNA polymerase II moves
through them
• They are also recognized by the presence of RNA
binding protein and processing enzymes
• CstF (Cleavage stimulation factor) and CPSF
(cleavage and polyadenylation specificity factor) travel
with the RNApolymerase II and transferred to 3’ end
processing seq on an RNA mol emerges from the
enzyme

Polyadenylation

• Subunits of CPSF are associated with
transcription factor TFIID
• During transcription initiation these subunits
moves on to RNA polymerase tail
• Once RNA mols comes from the polymerase
it will accompanied by the binding proteins to
form 3’endof mRNA. 1st RNA cleaved by this
• Next the enzyme called poly-A polymerase
adds, one time 200 nt A nt to the 3’end to
create a cut
• 5’ and 3’ end has been formed by ATP mol
• RNA Polymerase A does not require a RNA
template, hence not coded in the genome
• Poly A site is synthesized by addition of
Adenines on 3’ end
• Poly-A tail on mRNA now enters in the
cytosol and help to direct the synthesis of
proteins on ribosome

• After 3’ end of eukaryotes pre-mRNA mol have
been cleaved the RNA polymerase II while keep
transcribing
• Soon after polymerase looses its grip on RNA
template and termination a occurs
• Piece of RNA downstream of the cleavage site
is then degraded in the cell nucleus
• Two theories for RNA polymerase processivity
loss
1- transfer of 3’ end processing factors from RNA
polymerase to RNA causes a conformational
changes in the polymerase
2- Lack of 5’ cap of the RNA that arises form
polymerase might signals to eh enzyme to
terminate transcription

Transport of Eukaryote mRNA from the Nucleus
• The introns, broken RNA and cryptic sites are
dangerous to the cells
• That’s why mRNA is trnasferred to the cytosol for
translation
• Transport is done via nuclear pore complex for
complete processed mRNA
• Appropriate sets of proteins should be bound on
mRNA to be transported to the cytosol
• Nuclear Pore Complex
1- They are aqueous channels in the nuclear
membranes that directly connect nucleoplasm and
cytosol
2- Small mols < 50,000 daltons can diffuse freely
through them
3- Macromols signals cells for import and
exportpolymerase or mRNA respectively

Export-ready mRNA molecule

Nuclear Pore Complex
1- They are aqueous channels in the nuclear
membranes that directly connect nucleoplasm
and cytosol
2- Small mols < 50,000 daltons can diffuse freely
through them
3- Macromols signals cells for import and
exportpolymerase or mRNA respectively
4- Only useful RNA exported out via pores
5- hnRNPs (heterogenous nuclear ribonuclear
proteins, 30 of them in humans)
are present in abundant on the pre-mRNA
6- Some are useful in removing hairpin helices
from the RNA
7- Some pack RNA in long introns seq found in
genes of complex organism

8- Besides histones, hnRNP proteins are most
abundantin the cell nucleus
9- These proteins help to distinguished b/w
mature and unprocessed mRNA
10- Hoever hnRNP are reoved from the exon seq
proir binding of spliceosome components
11- They are destroyed later
12- mRNA with its 5’ end first enters in to the
cytosol with structural trnasitions

Noncoding RNAs synthesis: rRNA
• Few % of dry cell weight is RNA
•The most abundant RNA in the cell is rRNA ie
80%
• 3-5 % mRNA
• RNA polymerase I (structually similar to II)
produces rRNA
• RNA polymerase I have no C-terminal ie why
they are neither capped or polyadenylated
• Ribosome are final gene products and a growing
cell must synthesize approx. 10 million copies of
each type of rRNA in each cell generation to
construct its 10 million ribosomes
• This is done by the presence of enough rRNA
genes for rRNAs.
• Ecoli need 7 copies of its rRNA genes copies per
haploid genome, on 5 chromosomes

Eukaryotic rRNAs
• 4 types of rRNAs, one on each copy of ribosome
• 3 of four are 18S, 5.8S and 28S
• They are synthesized from a chemically modified
and cleaved process generating single large
precursor rRNA
• 5.8S is synthesized from a separate cluster of
genes by a different polymerase
• RNA polymerase III does not require chemical
modification and transcribed separately
• Many chemical modifications occur in the 13,000-
nucleotide-long precursor r RNA before the rRNA
are cleaved out of it and assembled into ribosomes
• Chemical modifications includes 100 methylations
of the 2’OH positions on nt sugars and 100
isomerizations of uridine nt to pseudouridine

Modifications:
• It is made at specific position in the prescursor rRNA
• These positions are specified by several hundred “guide
RNAs” which locate themselves via bp to the precursor
rRNA thereby move RNA-modifying enzyme to the
appropriate position
• Other guide RNAs promote cleavage of the precursor
rRNAs in to the mature rRNAs probably by causing
conformational changes in the precurosor rRNA
• They all belong to theRNA class of small nucleolar RNAs
(snoRNAs)
• Many snoRNA are encoded in the introns of other genes
specially those encoding ribosomal proteins
• They are therefore synthesized by RNA polymerase II and
processed from excised intron sequences

Nucleolus: A Ribosomal-Producing Factory
• Nucleolus is the rRNA processing site and their
assemblky into ribosome
• It is large aggregate of macromolecules, including
the rRNA genes themselves, precursor RNAs,
mature rRNAs, rRNA processing enzymes,
snoRNPs, ribosomal protein subunits and partly
assembled ribosomes
• It is the site where other RNAs are produced and
other RNA-protein complexex are assembled
• An eg: The U6 snRNA is chemically modified by
snoRNAs in the nucleolous before its final assembly
there into the U6 snRNP
• Telomerase and signal recognition particle are
said to be synthesized in nucleolus
• tRNA are also processed in here
• Nucleolus size changes with the type of cells

The Nucleus
• Contains GEMS (Gemini of coiled bodies) and
speckles (interchromatin granule clusters)
• These structures are highly dynamic bc of tight
association of RNA-protein
• GEMS and speckles are paired in the nucleus
•This is the site where snRNAs and snoRNAs
undergo their final modifications and assemble with
proteins
• RNA and proteins for snRNPs synthesis are partly
assembled in cytoplasm but are transported in to the
nucleus for final modification
• They are the Cajal/GEMS site where snRNPs are
recycled and their RNAs are “reset” after
rearrangements that occur during splicing of pre-
mRNA

The Nucleus
• Interchromatin granule clusters have been proposed
to be stock piles of fully mature snRNPs that are ready
to be used in splicing of pre-mRNA
• GEMS contain SMN (survival motor neurons)
proteins. Mutations in GEMS gene encoding for this
protein can cause the spinal muscular atrophy
• In this disease subtle defects in snRNPs assembly
and subsequent splicing of pre-mRNA takes place
• RNA splicing take place at various location in
chromosome bc splicing is co-transcriptional
• At Interphase when chromosomes occupy discrete
territories in the nucleus, then transcription and pre-
mRNA splicing takes place
• Chromosomes at interphase and can be located via
gene expression
For e.g. Presence of transcriptionally silent regions of
interphase chromosomes are often associated with
nuclear envelop where the conc. of heterochromatin
components is believed to be esp. high

The Nucleus
• But when the same region become
transcriptionally active, they relocate towards the
interior of the nucleus, which is richer in the
components required for mRNA synthesis
• Mammalian cells express 15,000 genes so
transcription and RNA splicing must take place
at several thousands sites in the nucleus
• Nucleus is seems to be very dynamic and
divided in to subdomains, with snRNPs,
snoRNPs and other nuclear components moving
between them according to the need of the cell

Lecture.6 DNA Transcription

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Lecture.6 DNA Transcription