Kim-Lee B.

Domingo

Transcription

Transcription - The synthesis of RNA molecules using DNA strands as the templates so
that the genetic information can be transferred from DNA to RNA

Template strand - is the strand from which the RNA is actually transcribed. It is also
termed as antisense strand.

Coding strand - is the strand whose base sequence specifies the amino acid sequence
of the encoded protein. Therefore, it is also called as sense strand.

RNA (Ribonucleic Acid): Templete = single stranded, Substrate = NTP, Enzyme =RNA
Polymerase, Product = single stranded RNA, Base pair = A-U, C-T.
Transcription Processes and Factors

Steps Prokaryotes Eukaryotes

RNA Polymerase II
1. Histone  HAT (histone
Acetylation acetyltransferase) sub-unit
will acetylate the N -terminal
histone tails of H2A, H2B,
H3 (histone 3) and
H4(histone 4) or either of the
histones. It will trigger
nucleosome disrupt near the
TATA elements for efficient
transcrption.
 HAT function is
enzymatically by
transferring acetyl
group from acetyl co-
enzyme A(acetyl-
 CoA) to the amino
terminal group of
cetain lysine side
chains in the histone’s
basic N-terminal tail
region.
 Lysine acetylation
neutralize the part of
a tail’s region positive
charge to weakens
the histone-DNA or
nucleosome-
nucleosome
interactions.

2. Transcriptio
n initiation Pribnow  mRNA
RNA box Hogness-Goldberg box
polymerase (promoter
binds to sequence which
DNA at a lies directly
specific upstream of the
sequence of transcribed
nucleotides sequence of the
called the gene, signals for
promoter. the initiation of
-The transcription)
promoter
contains
specific DNA
sequences
that act as
points of
attachment
for the RNA
polymerase.

Recognition of The RNA RNA polymerase II-produce mRNA-

gene promoters. polymerase nucleoplasm
holoenzyme
(core enzyme+
 factor)
initially binds to
the promoter at
nucleotide
 positions
Formation of a - TBP subunit (TFIID) recognition of TATA
closed promoter Sigma factors box
complex - The  factor is - TFIIA and TFIIB(accurate positioning of
preinitiation primarily RNA polymerase at the start of
complex involved in transcription) enters the complex, which
recognition of stabilize the DNA-TFIID complex and
gene promoters. recruit Pol II in association with TFIIF and
s70-General additional transcription factors. TFIIF
“housekeeping” serves as the bridge between the TATA-
genes bound TBP and polymerase. One of the
last transcription factors to be recruited to
S-General the preinitiation complex is TFIIH, which
stress regulator plays an important role in promoter
genes (> 70 melting and escape
genes)

32-Heat shock
proteins:
chaperone
proteins and
proteases that
fold or degrade
damaged
proteins

E-Genes that
restore envelope
integrity

Promoter melting promoter melting requires hydrolysis of

and open complex ATP and is mediated by TFIIH.
formation While the upstream promoter DNA is held
in a fixed position by TFIID, TFIIH pulls
downstream double-stranded DNA into the
cleft of the polymerase, driving the
separation of DNA strands and the
transition of the preinitiation complex from
the closed to open state. TFIIB aids in
open complex formation by binding the
melted DNA and stabilizing
the transcription bubble.
Addition of energy F (28)- polymerization reaction in the absence of
to begin Flagellum a primer.
transcription assembly and
Abortive initiation - polymerase may
chemotaxis
terminate prematurely and release a short,
 truncated transcript

FecI-Transport Promoter escape –

machinery for
When a transcript attains the threshold
iron citrate
length of ten nucleotides, it enters the
uptake
RNA exit channel.The polymerase breaks
its interactions with the promoter elements
and any regulatory proteins associated
with the initiation complex that it no longer
needs.Promoter escape in eukaryotes
requires ATP hydrolysis and, in the case
of Pol II-phosphorylation of the CTD.
Meanwhile, the transcription bubble
collapses down to 12-14 nucleotides,
providing kinetic energy required for the
escape.

54-Metabolism
of alternative
nitrogen sources
The sigma factor
stimulates tight
binding of RNA
polymerase to
the promoter.
2. Elongation conformational change in the core enzyme
Addition of RNA
nucleotides
 Addition Double stranded DNA that enters from the
of front of the enzyme is unzipped to avail
ribonucle the template strand for RNA synthesis. For
otides by every DNA base pair separated by the
RNA Pol advancing polymerase, one hybrid
to the 3'- RNA:DNA base pair is immediately
end of the formed. DNA strands and nascent RNA
RNA chain exit from separate channels; the two
 the RNA DNA strands reunite at the trailing end of
chain the transcription bubble while the single
grows in strand RNA emerges alone.
5'-3'
direction.
After
adding 10
nucleotid
es, σ
dissociate
 s.

RNA
Polymerase
subunits: 2α, β,
β’ - fully capable
of catalyzing the
polymerization
of NTPs into
RNA ; σ-
required to
identify the
correct sites for
transcription
initiation

 NusG –
increases
the
elongation
rate by
inhibiting the
backtracking

Pausing and  GreA and

proofreading GreB – in  TFIIS- stimulates an inherent
E. coli ribonuclease activity in the
suppress polymerase, allowing the removal of
RNAP misincorporated bases through limited
pausing local RNA degradation
 ELL- an RNA Pol II elongation factor
that facilitates RNA polymerase II
pause site entry and release
 ELL and TFIIS stimulate the rate of
elongation by limiting the length of
time that polymerase pauses.

Release of
the strand,  NELF (negative elongation factor)-
RNA mediates pausing of transcription in
capping, collaboration with DSIF (DRB-
sensitivity-inducing factor containing
SPT4/SPT5)
  The blockage is released once
the polymerase receives an
activation signal, such as the
phosphorylation of Ser-2 of
CTD tail by P-TEFb

 P-TEFb,- phosphorylates the second

residue (Ser-2) of the CTD repeats
(YSPTSPS) of the bound Pol II.
 phosphorylates and activates
SPT5 and TAT-SF1.
 SPT5 is a universal
transcription factor that
helps recruit 5'-capping
enzyme to Pol II with a
CTD phosphorylated at
Ser-5.
 TAT-SF1 recruits
components of the RNA
splicing machinery to the
Ser-2 phosphorylated
CTD.
 helps suppress transient
pausing of polymerase when it
encounters certain sequences
immediately following initiation

 7-methyl guanosine (7-MG) cap is

added to the 5’ end when the growing
RNA chain is fairly short (20-30 bases
in length).\
o The 7-MG cap is
“attached” by an unusual
5’-5’ triphosphate
linkage and serves to
protect the growing RNA
from degradation by
guanyltransferase and
methytansferase.
o nucleotide of the
nascent RNA is
converted to a
diphosphate by the 5_-
 triphosphatase, followed
by the fusion of a GMP
moiety to this same
nucleotide by
guanylyltransferase
activity
o methyltransferase adds
a methyl group to the N7
position of the GMP
moiety, completing the
cap structure
This “capping” is part of RNA processing in
eukaryotes

3. Termination Rho-  Pol ll – at the end of a gene, two

RNA polymerase independent protein complexes carried by the
releases the RNA termination - CTD, CPSF (cleavage and
polymer and “intrinsic polyadenylation specificity factor) and
detaches from the terminators” CSTF (cleavage stimulation factor),
DNA because they recognize the poly-A signal in the
cause transcribed RNA. Poly-A-bound CPSF
termination of and CSTF recruit other proteins to
transcription in carry out RNA cleavage and then
the absence of polyadenylation.
any external
factors Transcription by RNA Pol II is not really
terminated, in the sense that transcription
Rho-dependent continues for 1,000 - 2,000 bases after or
termination - downstream from the site that ultimately will
require the Rho become the 3’ end of the mature transcript.
protein; without The “functional” transcript actually results
it RNA from endonucleolytic cleavage of the primary
polymerase transcript.
continues  Cleavage occurs 10-30 bases
to transcribe downstream from the conserved
past the sequence AAUAAA.
terminator, a
process known Polyadenylation
as readthrough  After cleavage, an enzyme [poly(A)
polymerase] adds about 200 adenine
(A) bases to the 3’ends. This is called
polyadenylation or the addition of
poly-A tails.The function of poly-A tails
is to increase stability of the transcript
and to assist in transport of the mRNA
from the nucleus to the cytoplasm.
Post transcriptional
processes Splicing
 Eukaryotic transcripts possess
extra segments (introns or
intervening sequences). They
are removed by nucleases.
Ribozyme (an-RNA enzyme) is
a self splicing intron involved in
some of these reactions as well
as catalysingpolymerisation.

Splicing by the spliceosome

the U1 snRNP forms base pairs with the 5’

splice junction and the BBP (branch-point
binding protien) and U2AF (U2 auxiliary
factor) recognize the branch point site.
The U2 snRNP displays BBP and U2AF and
forms base pairs with the branch point site
consensus
The U4/U6-U5 “triple” snRNP enters the
reaction. In this triple snRNP, the U4 and U6
snRNA are held firmly together by the base-
pair interactions. Subsequent
rearrangements create the active site of the
spliceosome andposition the appropriate
portions of the pre-mRNA substrate for the
first phosphoryl-transferase reaction.
Several more rearrangements occur that
break apart the U4/U6 base pairs and allow
the U6 snRNP to dis[lace U1 at the 5’ splice
junction to form the active site for the second
phosphoryl-transferase reaction, which
completes the splice.

Eukaryotic Transcription
Steps RNA Polymerase I RNA Polymerase III
1. Transcription RNA polymerase I-  
Initiation produce rRNA located at RNA polymerase III- 5S
(Promotor) Nucleolus rRNA/tRNAs- nucleoplasm
  Core element  Promoter elements of most
and UCE RNAPIII genes, including
(Upstream tRNAs and 5S rRNA, lie
Control Element) within the transcribed
region
 . tRNA genes - have two
conserved internal elements,
termed the A box and B box.
  5s RNA - has A and C box

2. Transcription - The promoter  tRNA

Initiation sequence which - tRNA gene promoters
is located at the consist of two separated
150bp upstream 10bp elements (Box A and
the transcription B) located downstream of
initiation site is the transcription site.
recognized by two - TF IIIC binds to the box B.
transcriptions - After binding to TFIIIC, Box
factors. A orients TFIIIC towards the
- This transcription start site
factors are UBF - TFIIIC causes correct
and SL1 (sub-unit positioning of TFIIIB (Pre-
of TFIB) which Initiation Complex) and the
bind to the RNA binding protein Nab2
promoter. facilitates the interaction.
- Then the TFIB Which then recruits RNA
binds to the core Pol-III
region. - This DNA/TFIIIB (General
- Then it recruits Transcription Factor-
the RNA IIIB) /Pol III initiation
Polymerase I to Complex is very stable and
the promoter to may pass through many
form initiation rounds of tRNA transcription
complex. - Once TFIIIB has bound,
then RNA Pol-III can bind
 UBF – Recognizes and initiate transcription in
both the UCE and the presence of
Core element. ribonucleoside
 SL1 – one sub-unit is triphosphates. Initiation
tata binding protien by RNA Polymerase-III does
not require hydrolysis of an
ATP.
- Once TFIIIB binds, TFIIIC c
an be removed without
affecting initiation by RNA
Polymerase-III.
  5s rRNA
- TF IIIA binds to the
intragenic (lying within the
transcribed DNA sequence)
5s rRNA, the C box.
- Follwed by the binding of
the TF IIIB and TFIIIC.
TFIIIA serves as a platform
that replaces the A and B
box for positioning. TFIIIC
in an rotation with respect
on the site of transcription.
- Once the TFIIIC is bound to
the TFIIIA-DNA complex,
the Assembly of the TFIIIB
proceeds.
- TFIIIC causes the correct
positioning of TFIIIB to form
Pre-Initiation complex which
then recruits RNA Pol. III.

 RNAPIII is a 17-subunit
enzyme that functions
together with 3 transcription
factors: TFIIIA, TFIIIB and
TFIIIC
 TFIIIA is a single protein and
is specific for transcription
only of the 5S rRNA
gene, RDN5.
 TFIIIB is composed of Brf1,
Bdp1 and TBP (TATA-
binding protein), which is
common to all eukaryotic
RNAPs.
 TFIIIC is a large, flexible six-
subunit complex that
recognizes the promoter
elements of all RNAPIII
transcription units.

Transcription - The presence of the tRNA

Elongation heterodimer Rpa36 - During transcription
 and Rpa34 in the RNA elongation, Nab2 remains
Pol. I responsible for associated with RNAPIII
the unwinding and and/or the nascent
repair of the rRNA transcript.
gene. - RNA Pol. III elongates at an
 Human - average of ~20 nt/s.
hPAF53/CAST - TFIIIC remains associated
ortholog of Rpa36 with the tDNA, possibly
and Rpa34 because it is not
- Elongation proceeds simultaneously displaced
upon binding of the from both the A and B boxes
DNA TopI
(Topoisomerase I) to
the initiation complex. 5s rRNA
Which involves in the - Using KMnO4 and
addition of the new diethylpricarbonate to detect
nucleotides to the the single standed region in
rRNA transcript. the DNA
- Melted bubbles form upon
binding of Pol. III
- Melted bubbles initiates
upstream of +1 and
propagates downstream
- Pol. III melts the DNA
around the initiation sitewith
the active participation of
TFIIIB
- As Pol. III progress bubble
of melted DNA moves with
it.

Transcription Ribosomal genes carry Pol III - termination signal consists

Termination short DNA sequence at of a stretch of 5-7 thymines (on the
their 3’ ends which nontemplate strand) located within
contain a Sall Site (Sal- 40bp downstream from the 3' end of
Box) and causes mature RNAs
termination. - The weak base - pairing of
o TTFI (transcription the interactions of oligo(dA)
Termination in the template stand and
Factor, RNA Pol. I) oligo(U) in the nascent
protein bind to this transcripts as principal
region and causes destabilizing signal
DNA binding which - The oligo (T) is then
causes the identified by the C53 and
cessation of RNA C37 sub-units of RNA Pol.
transcription and III.
the disassembly of - Then the C11 responsible
 the RNA Pol. I for cleaving its 3’ end.
complex
o 3’-end of the  Human cells- terminating at
transcript is the U – rich tract > 50 nt
cleaved, from the 3’-ends of tRNA
generating a large
primary rRNA -three subunits of RNA Pol. III
molecule that is important in Termination
further processed  C53 and C37 – form a
into the mature heterodimer and are
18S, 5.8S and 28S engaged of the termination
rRNAs. signal
 C11- required for RNA
Cleavage. Which is
responsible for 3’ RNA
cleavage during terminator
pausing.

Post transcriptional Cleavage Cleavage

processes - Larger RNA  tRNA
precursors are - Several nucleotides are
cleaved to form removed from the 5’ and 3’
smaller RNAs. ends of the tRNA precursor
- Primary transcript by endonuclease RNase P
of rRNA is 45S in and exonuclease RNase D
eukaryotes which respectively
is then cleave - Further post transcriptional
with the help of processing may include
the exonuclease addition of 5’CAA3’ to the 3’
to removed the end.
spacer sequence. - RNase D cleaved tRNA
- molecule in a reaction
Splicing catalyzed by tRNA
- rRNA undergo nucleotidyltransferase.
self splicing which - The enzyme binds to three
splice the ribonucleoside triphosphate
precursors in separate active
transcript (28s,
sites and forms
18s, 5.8s rRNA).
phosphodiester bonds
- This involves the
leading to synthesis of 5’
nucleophilic CCA 3’ sequence which is
attack at the 5’- attached to the 3’ end of the
splice site by the mature tRNA molecule.
the 3’-OH of an
exogenous  5s rRNA
guanosine
 cofactor. Surplus 5s RNA is degraded in
- This reaction the nucleus.
adds the Undergoes only minor
guanosine onto processing.
the 5’-end of the - Removal of the 10-15
intron and release nucleotides from the 3’ end.
the 5’ exon.
Splicing
tRNA
- splicing reactions the 3’
hydroxyl group of a
guanosine nucleotide or
nucleoside cofactor makes a
nucleophilic attack on the
phosphate of the
phosphodiester bond at the
exon intron junction forming
a new phosphodiester bond
with the 5’ end of the intron.
- The 3’ hydroxyl group of
the displaced exon now
similarly attacks the 3’ end
of the intron resulting in
removal of intron and
splicing of the exons
together

 5s rRNA –no splicing.

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