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Chapter 11 Transcription The biochemistry and molecular biology department of CMU

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Page 1: Transcriptionlibvolume1.xyz/biochemistry/bsc/semester5/... · –The prokaryotic RNA polymerase is a multiple-subunit protein of ~480kD. –Eukaryotic systems have three kinds of

Chapter 11

Transcription

The biochemistry and molecular

biology department of CMU

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The synthesis of RNA molecules using

DNA strands as the templates so that

the genetic information can be

transferred from DNA to RNA.

Transcription

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• Both processes use DNA as the

template.

• Phosphodiester bonds are formed in

both cases.

• Both synthesis directions are from 5´́́́

to 3´́́́.

Similarity between

replication and transcription

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replication transcription

template double strands single strand

substrate dNTP NTP

primer yes no

Enzyme DNA polymerase RNA polymerase

product dsDNA ssRNA

base pair A-T, G-C A-U, T-A, G-C

Differences between

replication and transcription

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Section 1

Template and Enzymes

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• The whole genome of DNA needs to

be replicated, but only small portion

of genome is transcribed in response

to the development requirement,

physiological need and

environmental changes.

• DNA regions that can be transcribed

into RNA are called structural genes.

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§§§§1.1 Template

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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G C A G T A C A T G T C5' 3'

3' C G T C A T G T A C A G 5' template strand

coding strand

transcription

RNAG C A G U A C A U G U C5' 3'

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• 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 is opposite.

• This feature is referred to as the

asymmetric transcription.

Asymmetric transcription

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5'

3'

3'

5'

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Organization of coding information in the adenovirus genome

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§§§§1.2 RNA Polymerase

• The enzyme responsible for the RNA

synthesis is DNA-dependent RNA

polymerase.

– 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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core enzymeholoenzyme

Holoenzyme

The holoenzyme of RNA-pol in E.coli

consists of 5 different subunits: αααα2 ββββ β′β′β′β′

ωωωωσσσσ.

ωωωω

β′β′β′β′

ββββ

αααα αααα

σσσσ

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subunit MW function

αααα 36512 Determine the DNA to be

transcribed

ββββ 150618 Catalyze polymerization

β′β′β′β′ 155613 Bind & open DNA template

σσσσ 70263 Recognize the promoter

for synthesis initiation

RNA-pol of E. Coli

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• Rifampicin, a therapeutic drug for

tuberculosis treatment, can bind

specifically to the ββββ subunit of RNA-

pol, and inhibit the RNA synthesis.

• RNA-pol of other prokaryotic

systems is similar to that of E. coli in

structure and functions.

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RNA-pol I II III

products 45S rRNA hnRNA

5S rRNA

tRNA

snRNA

Sensitivity

to Amanitin No high moderate

RNA-pol of eukaryotes

Amanitin is a specific inhibitor of RNA-pol.

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• Each transcriptable region is called

operon.

• One operon includes several structural

genes and upstream regulatory

sequences (or regulatory regions).

• The promoter is the DNA sequence that

RNA-pol can bind. It is the key point

for the transcription control.

§§§§1.3 Recognition of Origins

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5'

3'

3'

5'

regulatory sequences

structural gene

promotorRNA-pol

Promoter

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5'

3'

3'

5'-50 -40 -30 -20 -10 1 10

start -10

region

T A T A A T

A T A T T A

(Pribnow box)

-35 region

T T G A C A A A C T G T

Prokaryotic promoter

Consensus sequence

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Consensus Sequence

Frequency in 45 samples 38 36 29 40 25 30

37 37 28 41 29 44

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• The -35 region of TTGACA sequence

is the recognition site and the

binding site of RNA-pol.

• The -10 region of TATAAT is the

region at which a stable complex of

DNA and RNA-pol is formed.

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Section 2

Transcription Process

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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 requires 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.

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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 (αααα2ββ′σββ′σββ′σββ′σ) - DNA - pppGpN- OH 3′′′′

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• No primer is needed for RNA

synthesis.

• The σσσσ 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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b. Elongation

• The release of the σσσσ 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 of the nascent RNA strand.

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• RNA-pol, DNA segment of ~40nt and

the nascent RNA form a complex

called the transcription 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.

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Transcription bubble

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RNA-pol of E. Coli

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RNA-pol of E. Coli

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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 ρρρρ -dependent or ρρρρ -independent manner.

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The termination function of ρρρρ factor

The ρρρρ factor, a hexamer, is a ATPase

and a helicase.

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ρρρρ-independent termination

• The termination signal is a stretch of

30-40 nucleotides on the 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 structures to

terminate the transcription.

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RNA

5′′′′TTGCAGCCTGACAAATCAGGCTGATGGCTGGTGACTTTTTAGGCACCAGCCTTTTT... 3′′′′

DNA

UUUU...?

rplL protein

UUUU...?

5′′′′TTGCAGCCTGACAAATCAGGCTGATGGCTGGTGACTTTTTAGTCACCAGCCTTTTT... 3′′′′

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• The stem-loop structure alters the

conformation of RNA-pol, leading 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.

Stem-loop disruption

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§§§§2.2 Transcription of Eukaryotes

• 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.

a. Initiation

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structural geneGCGC CAAT TATA

intronexon exon

start

CAAT box

GCbox

enhancer

cis-acting element

TATA box (Hogness box)

Cis-acting element

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TATA box

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• RNA-pol does not bind the promoter

directly.

• 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.

Transcription factors

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TF for eukaryotic transcription

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• TBP of TFII D binds TATA

• TFII A and TFII B bind 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)

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Pre-initiation complex (PIC)

RNA pol II

TF II F

TBP TAF

TATADNA

TF II

ATF II

B

TF II E

TF II H

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• TF II H is of protein kinase activity to phosphorylate CTD of RNA-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.

Phosphorylation of RNA-pol

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• The elongation is similar to that of

prokaryotes.

• The transcription and translation do

not take place simultaneously since

they are separated by nuclear

membrane.

b. Elongation

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RNA-Pol

RNA-Pol

RNA-Pol

nucleosome

moving

direction

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• The termination sequence is AATAAA

followed by GT repeats.

• The termination is closely related to

the post-transcriptional modification.

c. Termination

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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 systems.

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• 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

§§§§3.1 Modification of hnRNA

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CH3

O

O OH

CH2

PO

O

O

N

NHN

N

O

NH2

AAAAA-OH

O

Pi

5'

3'

O

OHOH

H2CN

HNN

N

O

H2N O P

O

O

O P

O

O

O P

O

O

5'

a. Capping at the 5′′′′- end

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

DNA template. ⇒⇒⇒⇒ The tailing process

dose not depend on the template.

• The tailing process occurs prior to the

splicing.

• The tailing process takes place in the

nuclei.

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The matured mRNAs are much shorter than

the DNA templates.

DNA

mRNA

c. mRNA splicing

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

L 1 2 3 4 5 6 77 700 bp

The structural genes are composed of

coding and non-coding regions that

are alternatively separated.

Split gene

E A B C D F G

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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

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Splicing mechanism

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lariat

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U pA G pU5' 3'5'exon 3'exon

intron

pG-OH

pGpA

G pU 3'U5' OH

first transesterification

Twice transesterification

second transesterification

U5' pU 3'

pGpA

GOH

5'

3'

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• Taking place at the transcription

level

• One gene responsible for more than

one proteins

• Significance: gene sequences, after

post-transcriptional modification,

can be multiple purpose

differentiation.

d. mRNA editing

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Different pathway of apo B

Human apo B

gene

hnRNA (14 500 base)

liver

apo B100

((((500 kD)))) intestine

apo B48

((((240 kD))))

CAA to UAA

At 6666

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§§§§3.2 Modification of tRNA

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tRNA precursor

RNA-pol III

TGGCNNAGTGC GGTTCGANNCC

DNA

Precursor transcription

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RNAase P

endonuclease

Cleavage

ligase

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tRNA nucleotidyl

transferase

ATP ADP

Addition of CCA-OH

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Base modification

((((1111))))

((((1111))))

((((3333))))

((((2222))))

((((4444))))

1. Methylation

A→mA, G→mG

2. Reduction

U→DHU

3. Transversion

U→ψ

4. Deamination

A→I

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§§§§3.3 Modification of rRNA

• 45S transcript in nucleus is the

precursor of 3 kinds of rRNAs.

• The matured rRNA will be assembled

with ribosomal proteins to form

ribosomes that are exported to

cytosolic space.

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rRNA

transcription

splicing

45S-rRNA

18S-rRNA5.8S and 28S-rRNA

28S5.8S18S

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• The rRNA precursor of tetrahymena

has the activity of self-splicing (1982).

• The catalytic RNA is called ribozyme.

• Self-splicing happened often for

intron I and intron II.

§§§§3.4 Ribozyme

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• Both the catalytic domain and the

substrate locate on the same

molecule, and form a hammer-head

structure.

• At least 13 nucleotides are conserved.

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Hammer-head

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• Be a supplement to the central

dogma

• Redefine the enzymology

• Provide a new insights for the origin

of life

• Be useful in designing the artificial

ribozymes as the therapeutical

agents

Significance of ribozyme

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Artificial

ribozyme

• Thick lines: artificial ribozyme

• Thin lines: natural ribozyme

• X: consensus sequence

• Arrow: cleavage point