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Transcription

RNA
• RNA can both store information (like DNA) 
and catalyze chemical reactions (like 
proteins).  
t i )

• One theory for the origin of life has it 
starting out as RNA only, then adding DNA 
and proteins later.  

• Recently it has been found that very small 
RNA molecules are involves in gene 
regulation.

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RNA vs. DNA

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Central dogma of Molecular Biology
• describes the way genetic information is
expected
t d to
t be
b transferred
t f d in
i a single
i l direction
di ti
through a biological system

Transcription

The synthesis of RNA

molecules using DNA
strands as the
templates so that the
genetic information
can beb t
transferred
f d
from DNA to RNA.

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• The whole genome of DNA needs to

be replicated, but only small portion
of genome is transcribed in response
t the
to th development
d l t requirement,
i t
physiological need and
environmental changes.
• DNA regions that can be transcribed
into RNA are called structural genes
genes.

The template strand is the

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

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55' GCAGTACATGTC 3' coding

3 g
strand
3' CGTCATGTACAG 5' template
strand

transcription

5' GCAGUACAUGUC 3' RNA

Asymmetric transcription
• Only the template strand is used for the
transcription, but the coding strand is
not.
• Both strands can be used as the
templates.
• The transcription direction on different
strands
t d is
i opposite.
it
• This feature is referred to as the
asymmetric transcription.

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5' 3'
3' 5'

1.2 RNA Polymerase

• The enzyme responsible for the RNA
synthesis
– The prokaryotic RNA polymerase is a
multiple-subunit protein of ~480kD.
– Eukaryotic systems have three kinds of
RNA polymerases, each of which is a
multiple-subunit protein and responsible
for transcription of different RNAs.

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Holoenzyme

The holoenzyme of RNA-pol in E.coli

consists of 5 different subunits: 2  
.



h oloen
l
core zy m e
enzyme  



RNA-pol of E. Coli

subunit MW function
Determine the DNA to be
 36512
transcribed

 150618 Catalyze polymerization

 155613 Bind & open DNA template

Recognize the promoter
 70263
for synthesis initiation

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• Rifampicin, a therapeutic drug for

tuberculosis treatment,, can bind
specifically to the  subunit of RNA-
pol, and inhibit the RNA synthesis.
• RNA-pol of other prokaryotic
systems is similar to that of E.
E coli in
structure and functions.

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RNA-pol of eukaryotes

RNA-pol I II III

5S rRNA
products 45S rRNA hnRNA tRNA
snRNA

S iti it
Sensitivity
No high moderate
to Amanitin

Amanitin is a specific inhibitor of RNA-pol.

§1.3 Recognition of Origins

• Each transcriptable region is
called operon.
• One operon includes several
structural genes and upstream
regulatory sequences
• The promoter is the DNA
sequence that RNA-pol
RNA pol can
bind. It is the key point for the
transcription control.

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Promoter

regulatory
structural gene
sequences
5' 3'
RNA-pol
promoter
3' 5'

Prokaryotic promoter

5' 3'
-50 -40 -30 -20 -10 1 10
3' 5'
-35
region -10 start
TTGACA region
AACTGT
TATAAT
ATATTA
(Pribnow box)

Consensus sequence

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Consensus Sequence

Frequency in 45 samples 38 36 29 40 25 30
37 37 28 41 29 44

• The -35 region of TTGACA sequence is

th recognition
the iti site
it and
d th
the bi
binding
di site
it
of RNA-pol.
• The -10 region of TATAAT is the region
at which a stable complex of DNA and
RNA pol is formed
RNA-pol formed.

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S ti
Section 2

Transcription Process

General concepts

• Three phases: initiation, elongation,

and termination.
• The prokaryotic RNA-pol can bind to
the DNA template directly in the
transcription process.
• The eukaryotic RNA-pol
RNA pol requires co
co-
factors to bind to the DNA template
together in the transcription process.

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§2.1 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.

a. 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.

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• 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 RNA-
pol once the first 3,5 phosphodiester
bond is formed.
• The core enzyme moves along the
DNA template to enter the elongation
phase.

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http://cnx.org/content/m44523/latest/?colle
ction=col11448/latest

b. 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
OH of the nascent RNA strand.
strand
(NMP)n + NTP (NMP)n+1 + PPi
elongated
RNA strand substrate RNA strand

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• RNA-pol, DNA segment of ~40nt and

the nascent RNA form a complex
called the transcription bubble.
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.

Synthesis of an RNA Transcript ‐ Elongation
• RNA polymerase synthesizes a single strand of RNA against the DNA template 
strand (anti‐sense strand), adding nucleotides to the 3’ end of the RNA chain
• As RNA polymerase moves along the DNA it continues to untwist the double 
helix, exposing about 10 to 20 DNA bases at a time for pairing with RNA 
nucleotides

Elongation Non-template
strand of DNA
RNA nucleotides

RNA
polymerase

T C C A A
A
3
3 end
U
A E G C A
5

T A G G T T

Direction of transcription
5 Template
(“downstream”)
strand of DNA

Newly made
RNA

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© 2002 by Bruce Alberts, Alexander Johnson, Julian Lewis, Martin Raff,

Keith Roberts, and Peter Walter.

Prokaryotic Transcription

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Simultaneous
transcriptions and
translation

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c. Termination

• The RNA-pol stops moving on the

DNA template. The RNA transcript
falls off from the transcription
complex.
• The termination occurs in either  -
dependent or  -independent manner.

The termination function of  factor

The  factor, a hexamer, is a ATPase

and a helicase.

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http://www.eplantscience.com/index/geneti
cs/expression_of_gene_protein_synthesis_
transcription_in_prokaryotes_and_eukaryot
es/termination_and_antitermination_of_mr
na_synthesis_in_prokaryotes.php

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-independent termination

• The termination signal is a stretch of

30 40 nucleotides
30-40 l tid on theth 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 (hairpin loop)
structures to terminate the
transcription.

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Stem-loop disruption
• The stem-loop structure alters the
conformation of RNA-pol, leading g to
the pause of the RNA-pol moving.
• Then the competition of the RNA-
RNA hybrid and the DNA-DNA hybrid
reduces the DNA-RNA hybrid stability,
and causes the transcription
complex dissociated.
• Among all the base pairings, the
most unstable one is rU:dA.

§2.2 Transcription of Eukaryotes

a. Initiation
• 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.

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Cis-acting element

cis-acting element
structural gene
GCGC CAAT TATA
exon intron exon

start
TATA box (Hogness box)

enhancer CAAT box

GC box

TATA box

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Transcription factors
• RNA-pol does not bind the promoter
directly.
y
• 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.

TF for eukaryotic transcription

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Pre-initiation complex (PIC)

• TBP of TFII D binds TATA

• TFII A and
d TFII B bind
bi d TFII D
• TFII F-RNA-pol complex binds TFII B
• TFII F and TFII E open the dsDNA
(helicase and ATPase)
• TFII H: completion of PIC

Pre-initiation complex (PIC)

RNA pol II

TF II F TF II E
TF II TBP TAF
TF II
A TATA B
TF II H DNA

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Phosphorylation of RNA-pol

• TF II H is of protein kinase activity to

phosphorylate CTD of RNA RNA-pol.
pol. (CTD
is the C-terminal domain of RNA-pol)
• Only the p-RNA-pol can move toward
the downstream, starting the
elongation phase.
• Most of the TFs fall off from PIC
during the elongation phase.

Transcription in eukaryotes
• Transcriptional activators bind to specific sequences
in DNA and help to attract RNA polymerase II to the
start point
i off transcription
i i

• transcription initiation requires the presence of a

protein complex known as the mediator

• Often requires the local recruitment of chromatin‐

modifying enzymes, including chromatin remodeling
complexes and histone acetylases

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• Activators: 
– gene regulatory proteins that help RNA 
polymerase, the general factors, and the mediator 
all to assemble at the promoter
– Attract ATP‐dependent chromatin‐remodeling
complexes and histone acetylases
• Mediators
– transcriptional activators
– allows the activator proteins to communicate 
ll th ti t t i t i t
properly with the polymerase II and with the 
general transcription factors.

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b. Elongation

• The elongation is similar to that of

prokaryotes
prokaryotes.
• The transcription and translation do
not take place simultaneously since
they are separated by nuclear
membrane.

nucleosome

RNA-Pol

moving
direction
di ti

RNA-Pol

RNA-Pol

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Transcription in Eukaryotes
• DNA topoisomerase
– enzymes that rapidly remove superhelical tension 
in DNA
• DNA gyrase
– uses the energy of ATP hydrolysis to pump 
supercoils continuously into the DNA, thereby 
maintaining the DNA under constant tension

c. Termination

• The termination sequence is AATAAA

followed by GT repeats.
• The termination is closely related to
the post-transcriptional modification.

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S ti
Section 3

Post-Transcriptional
Modification

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• The nascent RNA, also known as

primary transcript, needs to be
modified to become functional tRNAs,
rRNAs, and mRNAs.
• The modification is critical to
eukaryotic
k ti systems.
t

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§3.1 Modification of hnRNA

• Primary transcripts of mRNA are called as
heteronuclear RNA (hnRNA).
• hnRNA are larger than matured mRNA by
many folds.
• Modification includes
– Capping at the 5- end
– Tailing at the 3- end
– mRNA splicing
– RNA edition

a. Capping at the 5- end

OH OH
O
N
NH
O O O
O 5'
H2N N N H2C O P O P O P O CH2 N NH2
N
5' O
O O O
HN
N
O
CH3
O OH
Pi
O P O AAAAA OH3'
AAAAA-OH
O

m7GpppGp----

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• The 5- cap structure is found on

hnRNA too.  The capping process
occurs in nuclei.
• The cap structure of mRNA will be
recognized by the cap-binding protein
required for translation.
• The capping occurs prior to the
splicing.

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b. Poly-A tailing at 3 - end

• There is no poly(dT) sequence on the
template.  The tailing process
DNA template
dose not depend on the template.
• The tailing process occurs prior to the
splicing.
• Th
The tailing
t ili process takes
t k place
l in
i the
th
nuclei.

c. mRNA splicing

mRNA

DNA

The matured mRNAs are much shorter than

the DNA templates.

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Split gene

The
e st
structural
uctu a ge
genes
es a
are
e co
composed
posed o
of
coding and non-coding regions that
are alternatively separated.

7 700 bp
L 1 2 3 4 5 6 7
A B C D E F G

A~G no-coding region 1~7 coding region

Exon and intron

Exons are the coding sequences that

appear on split genes and primary
transcripts, and will be expressed to
matured mRNA.

Introns are the non-coding sequences

that are transcripted into primary
mRNAs, and will be cleaved out in the
later splicing process.

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mRNA splicing

Splicing mechanism

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lariat

Twice transesterification
intron

5'exon 3'exon
5' U pA G pU 3'

first transesterification
pG-OH
pGpA

5' UOH G pU 3'

second transesterification
5' pGpA

5' U pU 3'

GOH 3'

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RNA splicing is performed by 
Spliceosome
• Spliceosome,
– the large assembly of small nuclear RNA(snRNAs) 
and protein molecules that performs pre‐mRNA 
splicing in the cell.

d. mRNA editing

• Taking place at the transcription

level
• One gene responsible for more than
one proteins
• Significance: gene sequences, after
post-transcriptional
tt i ti l modification,
difi ti
can be multiple purpose
differentiation.

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Different pathway of apo B

Human apo B
gene

hnRNA (14 500 base)

CAA to UAA
At 6666
liver
apo B100
intestine
（500 kD）
apo B48
（240 kD）

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