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Chapter 11

Transcription

The biochemistry and molecularbiology department of CMU

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The synthesis of RNA molecules using

DNA strands as the templates so thatthe genetic information can betransferred from DNA to RNA.

Transcription

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Both processes use DNA as thetemplate.

Phosphodiester bonds are formed inboth cases.

Both synthesis directions are from 5 to 3 .

Similarity betweenreplication 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 betweenreplication and transcription

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Section 1

Template and Enzymes

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The wholegenome of DNA needs to

be replicated, but only small portionof genome is transcribed in response

to the development requirement,physiological need andenvironmental changes.

DNA regions that can be transcribedinto RNA are called structural genes.

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1.1 Template

The template strand is the strandfrom which the RNA is actuallytranscribed. It is also termed as

antisensestrand.

The coding strand is the strandwhose base sequence specifies the

amino acid sequence of the encodedprotein. Therefore, it is also called assensestrand.

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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' templatestrand

codingstrand

transcription

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

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Only the templatestrand is used for thetranscription, but the coding strand is

not.

Both strands can be used as thetemplates.

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 inthe adenovirus genome

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1.2 RNA Polymerase

The enzyme responsible for the RNAsynthesis is DNA-dependent RNApolymerase.

The prokaryotic RNA polymerase is amultiple-subunit protein of ~480kD.

Eukaryotic systems have three kinds of

RNA polymerases, each of which is amultiple-subunit protein and responsiblefor transcription of different RNAs.

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core enzymeholoenzyme

Holoenzyme

The holoenzyme of RNA-pol in E.coli

consists of 5 different subunits: 2

.

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subunit MW function

36512Determine the DNA to betranscribed

150618 Catalyze polymerization

155613 Bind & open DNA template

70263 Recognize the promoterfor synthesis initiation

RNA-pol of E. Coli

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Rifampicin, a therapeutic drug fortuberculosis 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. coliin

structure and functions.

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RNA-pol I II III

products 45S rRNA hnRNA

5S rRNA

tRNA

snRNA

Sensitivityto 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 regulatorysequences (or regulatory regions).

The promoter is the DNA sequence that

RNA-pol can bind. It is the key pointfor the transcription control.

1.3 Recognition of Origins

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5'

3'

3'

5'

regulatorysequences

structural gene

promotorRNA-pol

Promoter

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5'

3'

3'

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

start-10region

T A T A A TA T A T T A

(Pribnow box)

-35

region

T T G A C AA 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 thebinding site of RNA-pol.

The -10 region of TATAAT is theregion at which a stable complex ofDNA and RNA-pol is formed.

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Section 2

Transcription Process

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2.1 Transcription of Prokaryotes Initiation phase: RNA-pol recognizes

the promoter and starts thetranscription.

Elongation phase: the RNA strand iscontinuously growing.

Termination phase: the RNA-pol stopssynthesis and the nascent RNA isseparated from the DNA template.

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The first nucleotide on RNA transcript

is always purine triphosphate. GTP ismore often than ATP.

The pppGpN-OH structure remains on

the RNA transcript until the RNAsynthesis is completed.

The three molecules form a

transcription initiation complex.

RNA-pol ( 2 ) - DNA - pppGpN- OH 3

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No primer is needed for RNAsynthesis.

The subunit falls off from the RNA-pol once the first 3 ,5 phosphodiesterbond is formed.

The core enzyme moves along theDNA template to enter the elongationphase.

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b. Elongation The release of the subunit causes

the conformational change of thecore enzyme. The core enzyme slides

on the DNA template toward the 3 end.

Free NTPs are added sequentiallytothe 3 -OH of the nascent RNA strand.

(NMP)n + NTP (NMP)n+1 + PPi

RNA strand substrateelongated

RNA strand

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RNA-pol, DNA segment of ~40nt andthe nascent RNA form a complexcalled the transcription bubble.

The 3 segment of the nascent RNAhybridizes with the DNA template, andits 5 end extends out thetranscription 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 andtranslation

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c. Termination

The RNA-pol stops moving on theDNA template. The RNA transcript

falls off from the transcriptioncomplex.

The termination occurs in either -

dependent or -independent manner.

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The termination function of factor

The factor, a hexamer, is a ATPaseand a helicase.

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-independent termination

The termination signal is a stretch of30-40 nucleotides on the RNAtranscript, consisting of many GC

followed by a series of U.

The sequence specificity of thisnascent RNA transcript will formparticular stem-loop structures toterminate 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 theconformation of RNA-pol, leading tothe pause of the RNA-pol moving.

Then the competition of the RNA-RNA hybrid and the DNA-DNA hybridreduces the DNA-RNA hybridstability, 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 thespecific sequences on the DNA

template that regulate thetranscription of one or more genes.

a. Initiation

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structural gene

GCGC CAAT TATAintronexon exon

start

CAAT box

GC box

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 promoterdirectly.

RNA-pol II associates with six

transcription factors, TFII A - TFII H.

The trans-acting factors are theproteins that recognize and bind

directly or indirectly cis-actingelements 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 TAFTATADNA

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. (CTDis the C-terminal domain of RNA-pol)

Only the p-RNA-pol can move towardthe downstream, starting theelongation phase.

Most of the TFs fall off from PICduring 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 sincethey are separated by nuclearmembrane.

b. Elongation

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RNA-Pol

RNA-Pol

RNA-Pol

nucleosome

movingdirection

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Section 3

Post-Transcriptional

Modification

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The nascentRNA, also known asprimary transcript,needs to bemodified to become functional tRNAs,

rRNAs, and mRNAs.

The modification is critical toeukaryotic systems.

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Primary transcripts of mRNA are called asheteronuclear 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----

ppp5'NpNp

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ppp NpNp

pp5'NpNp

GTP

PPi

G5'ppp5'NpNp

methylating at G7

methylating at C2' of the

first and second

nucleotides after G

forming 5'-5'

triphosphate group

removing

phosphate group

m7GpppNpNp

m7Gpppm2'Npm2'Np

Pi

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The matured mRNAs are much shorter thanthe 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 ofcoding and non-coding regions that

are alternatively separated.

Split gene

EA B C D F G

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Exon and intronExons are the coding sequences thatappear on split genes and primarytranscripts, and will be expressed to

matured mRNA.

Introns are the non-coding sequencesthat are transcripted into primary

mRNAs, and will be cleaved out in thelater splicing process.

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mRNA splicing

S li i h i

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Splicing mechanism

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lariat

Twice transesterification

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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'

d RNA diti

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Taking place at the transcriptionlevel

One gene responsible for more than

one proteins

Significance: gene sequences, afterpost-transcriptional modification,can be multiple purposedifferentiation.

d. mRNA editing

Different pathway of apo B

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Different pathway of apo B

Human apo Bgene

hnRNA (14 500 base)

liver

apo B100500 kD

intestine

apo B48

240 kD

CAA to UAAAt 6666

3 2 Modification of tRNA

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3.2 Modification of tRNA

Precursor transcription

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tRNA precursor

RNA-pol III

TGGCNNAGTGC GGTTCGANNCC

DNA

Precursor transcription

Cleavage

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RNAase P

endonuclease

Cleavage

ligase

Addition of CCA OH

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tRNA nucleotidyl

transferase

ATP ADP

Addition of CCA-OH

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3 3 Modification of rRNA

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3.3 Modification of rRNA

45S transcript in nucleus is theprecursor of 3 kinds of rRNAs.

The matured rRNA will be assembledwith ribosomal proteins to formribosomes that are exported to

cytosolic space.

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rRNA

transcription

splicing

45S-rRNA

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

28S5.8S18S

3 4 Ribozyme

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The rRNA precursor of tetrahymenahas the activity of self-splicing (1982).

The catalytic RNA is called ribozyme.

Self-splicing happened often forintron I and intron II.

3.4 Ribozyme

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Both the catalytic domain and thesubstrate locate on the same

molecule, and form a hammer-headstructure.

At least 13 nucleotides are conserved.

Hammer head

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Hammer-head

Significance of ribozyme

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Be a supplement to the centraldogma

Redefine the enzymology

Provide a new insights for the originof life

Be useful in designing the artificialribozymes as the therapeuticalagents

Significance of ribozyme

Artificial

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Artificialribozyme

Thick lines:artificial ribozyme

Thin lines:natural ribozyme

X: consensussequence

Arrow: cleavage

point