11

TranscriptionTranscription

The biochemistry and molecular biology department of CMU

22

DNA

mRNA

Transcription

IntroductionIntroduction

The Central Dogma The Central Dogma of Molecular Biologyof Molecular Biology

Cell

Polypeptide(protein)

TranslationRibosome

33

MAJOR CLASSES OF RNAMAJOR CLASSES OF RNA

rRNA (Ribosomal RNA) 80% rRNA (Ribosomal RNA) 80% V.Stable.V.Stable.

mRNA (messenger RNA)2-5% mRNA (messenger RNA)2-5% Unstable.Unstable.

tRNA ( Transfer RNA) 15% V.Stable.tRNA ( Transfer RNA) 15% V.Stable. snRNA (Small nuclear RNA) 1% snRNA (Small nuclear RNA) 1%

V.Stable.V.Stable. miRNA (Micro RNA) 1% Stable.miRNA (Micro RNA) 1% Stable.

44

HOW RNA POLYMERASE FIND HOW RNA POLYMERASE FIND CORRECT SITECORRECT SITE

E.coli 4X10E.coli 4X1033 Transcription sites in Transcription sites in 4+106 bp long DNA.4+106 bp long DNA.

Humans 105 transcription sites in Humans 105 transcription sites in 3X103X1099 bp of DNA. bp of DNA.

How efficiently the entire genome is How efficiently the entire genome is scanned for the right gene unit for scanned for the right gene unit for the start of transcription.the start of transcription.

55

Pyrimidines

NH2

O

N

N NH

N

Guanine

N

N

Adenine

N

N

NH2

N O

NH2

N O

NH2

NCytosine

Uracil(RNA)CH3

N ON

O

NH

N ON

O

NH

Thymine(DNA)

Purines

©1998 Timothy G. Standish

Two Families of BasesTwo Families of Bases

66

DNA

Cytoplasm

Nucleus

Eukaryotic TranscriptionEukaryotic Transcription

ExportG AAAAAA

RNA

Transcription

Nuclear pores

G AAAAAA

RNAProcessing

mRNA

77 OH

OCH2

Sugar

HOH

A NucleotideA Nucleotide

NH2

N

N N

N

BaseP

O

OH

HO O

Phosphate

©1998 Timothy G. Standish

88

The synthesis of RNA molecules using DThe synthesis of RNA molecules using DNA strands as the templates so that the gNA strands as the templates so that the genetic information can be transferred froenetic information can be transferred from DNA to RNA.m DNA to RNA.

Transcription

99

ObjectivesObjectives

Understand the structure of RNA Understand the structure of RNA polymerasespolymerases

Understand the phases of the Understand the phases of the transcription cycletranscription cycle

Understand the differences between Understand the differences between transcription and replicationtranscription and replication

1010

Both processes use DNA as the tempBoth processes use DNA as the template.late.

Phosphodiester bonds are formed in Phosphodiester bonds are formed in both cases. both cases.

Both synthesis directions are from 5´ Both synthesis directions are from 5´ to 3´. to 3´.

Similarity between Similarity between replication and transcriptionreplication and transcription

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replicationreplication transcriptiontranscription

templatetemplate double strandsdouble strands single strandsingle strand

substratesubstrate dNTPdNTP NTPNTP

primerprimer yesyes no no

EnzymeEnzyme DNA polymeraseDNA polymerase RNA polymeraseRNA polymerase

productproduct dsDNAdsDNA ssRNAssRNA

base pairbase pair A-A-TT, G-C, G-C A-A-UU, T-A, G-C, T-A, G-C

Differences between Differences between replication and transcriptionreplication and transcription

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1313

The whole genome of DNA needs to The whole genome of DNA needs to

be replicated, but only small portion be replicated, but only small portion of genome is transcribed in response of genome is transcribed in response to the development requirement, to the development requirement, physiological need and physiological need and environmental changes. environmental changes.

DNA regions that can be transcribed DNA regions that can be transcribed into RNA are called structural genes.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 The enzyme responsible for the RNA synthesis is DNA-dependent RNA polsynthesis is DNA-dependent RNA polymerase.ymerase.The prokaryotic RNA polymerase is a mThe prokaryotic RNA polymerase is a m

ultiple-subunit protein of ~480kD. ultiple-subunit protein of ~480kD.

Eukaryotic systems have three kinds of Eukaryotic systems have three kinds of RNA polymerases, each of which is a mRNA polymerases, each of which is a multiple-subunit protein and responsible fultiple-subunit protein and responsible for transcription of different RNAs. or transcription of different RNAs.

2020

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The shape of each enzyme resembles a crab claw.

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core enzymeholoenzyme

Holoenzyme

The holoenzyme of RNA-pol in The holoenzyme of RNA-pol in E.coliE.coli con con

sists of 5 different subunits: sists of 5 different subunits: 22 ..

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subunitsubunit MWMW functionfunction

3651236512 Determine the DNA to be Determine the DNA to be transcribedtranscribed

150618150618 Catalyze polymerizationCatalyze polymerization

155613155613 Bind & open DNA templateBind & open DNA template

7026370263Recognize the promoterRecognize the promoter

for synthesis initiationfor synthesis initiation

RNA-pol of E. Coli

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Rifampicin, a therapeutic drug for tuRifampicin, a therapeutic drug for tuberculosis treatment, can bind specifberculosis treatment, can bind specifically to the ically to the subunit of RNA-pol, an subunit of RNA-pol, and inhibit the RNA synthesis. d inhibit the RNA synthesis.

RNA-pol of other prokaryotic systemRNA-pol of other prokaryotic systems is similar to that of s is similar to that of E. coliE. coli in structu in structure and functions. re and functions.

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RNA-polRNA-pol II IIII IIIIII

productsproducts 45S rRNA45S rRNA hnRNAhnRNA

5S rRNA5S rRNA

tRNAtRNA

snRNAsnRNA

Sensitivity tSensitivity to Amanitino Amanitin NoNo highhigh moderatemoderate

RNA-pol of eukaryotes

Amanitin is a specific inhibitor of RNA-pol.

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Each transcriptable region is called opeEach transcriptable region is called ope

ron.ron.

One operon includes several structural One operon includes several structural genes and upstream regulatory sequengenes and upstream regulatory sequences (or regulatory regions). ces (or regulatory regions).

The promoter is the DNA sequence that The promoter is the DNA sequence that RNA-pol can bind. It is the key point foRNA-pol can bind. It is the key point for the transcription control. r the transcription control.

§1.3 Recognition of Origins

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5'

3'

3'

5'

regulatory sequences structural gene

promotorRNA-pol

Promoter

2828

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 SequenceConsensus 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 The -35 region of TTGACA sequence is the recognition site and the bindinis the recognition site and the binding site of RNA-pol.g site of RNA-pol.

The -10 region of TATAAT is the regiThe -10 region of TATAAT is the region at which a stable complex of DNA on at which a stable complex of DNA and RNA-pol is formed.and RNA-pol is formed.

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Transcription ProcessTranscription Process

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General concepts

Three phases: initiation, elongation, Three phases: initiation, elongation, and termination. and termination.

The prokaryotic RNA-pol can bind to The prokaryotic RNA-pol can bind to the DNA template directly in the tranthe DNA template directly in the transcription process. scription process.

The eukaryotic RNA-pol requires co-fThe eukaryotic RNA-pol requires co-factors to bind to the DNA template toactors to bind to the DNA template together in the transcription process. gether in the transcription process.

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§2.1 Transcription of Prokaryotes

Initiation phase: RNA-pol Initiation phase: RNA-pol recognizes the precognizes the promoter and starts the transcription. romoter and starts the transcription.

Elongation phase: the RNA strand is cElongation phase: the RNA strand is continuously growing. ontinuously growing.

Termination phase: the RNA-pol stops Termination phase: the RNA-pol stops synthesis and the nascent RNA is sepasynthesis and the nascent RNA is separated from the DNA template. rated from the DNA template.

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a. Initiation

RNA-pol recognizes RNA-pol recognizes the TTGACA regiothe TTGACA region, and slides to the TATAAT region, then, and slides to the TATAAT region, then n opens the DNA duplex.opens the DNA duplex.

The unwound region is about 17The unwound region is about 171 bp. 1 bp.

3535

The first nucleotide on RNA transcript The first nucleotide on RNA transcript is always purine triphosphate. GTP is is always purine triphosphate. GTP is more often than ATP. more often than ATP.

The pppGpN-OH structure remains on The pppGpN-OH structure remains on the RNA transcript until the RNA syntthe RNA transcript until the RNA synthesis is completed. hesis is completed.

The three molecules form a transcriptiThe three molecules form a transcription initiation complex. on initiation complex.

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

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No primer is needed for RNA synthesiNo primer is needed for RNA synthesis. s.

The The subunit falls off from the RNA-p subunit falls off from the RNA-pol once the first 3ol once the first 3,5,5 phosphodiester phosphodiester bond is formed. bond is formed.

The core enzyme moves along the DNThe core enzyme moves along the DNA template to enter the elongation phA template to enter the elongation phase. ase.

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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 of the nascent RNA strand.

3838

• RNA-pol, DNA segment of ~40nt and t

he 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

4040

RNA-pol of E. Coli

4141

RNA-pol of E. Coli

4242

4343

4444

Simultaneous transcriptions and

translation

4545

c. Termination

The RNA-pol stops moving on the DNThe RNA-pol stops moving on the DNA template. A template. The RNA transcript falls oThe RNA transcript falls off from the transcription complex. ff from the transcription complex.

The termination occurs in either The termination occurs in either -de -dependent or pendent or -independent manner. -independent manner.

4646

The termination function of factor

The The factor,factor, a hexamer, is a a hexamer, is a ATPaseATPase an and a d a helicasehelicase. .

4747

-independent termination-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

5TTGCAGCCTGACAAATCAGGCTGATGGCTGGTGACTTTTTAGGCACCAGCCTTTTT... 3 DNA

UUUU...…

rplL proteinrplL protein

UUUU...…

5TTGCAGCCTGACAAATCAGGCTGATGGCTGGTGACTTTTTAGTCACCAGCCTTTTT... 3

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5050

The stem-loop structure alters the coThe stem-loop structure alters the co

nformation of RNA-pol, leading to the nformation of RNA-pol, leading to the pause of the RNA-pol moving.pause of the RNA-pol moving.

Then the competition of the RNA-RNThen the competition of the RNA-RNA hybrid and the DNA-DNA hybrid reA hybrid and the DNA-DNA hybrid reduces the DNA-RNA hybrid stability, duces the DNA-RNA hybrid stability, and causes the transcription compleand causes the transcription complex dissociated. x dissociated.

Among all the base pairings, the mosAmong all the base pairings, the most unstable one is rU:dA. t unstable one is rU:dA.

Stem-loop disruptionStem-loop disruption

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§2.2 Transcription of Eukaryotes

Transcription initiation needs promotTranscription initiation needs promoter and upstream regulatory regions. er and upstream regulatory regions.

The cis-acting elements The cis-acting elements are the speciare the specific sequences on the DNA template tfic sequences on the DNA template that regulate the transcription of one hat regulate the transcription of one or more genes. or more genes.

a. Initiation

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structural geneGCGC CAAT TATA

intronexon exon

start

CAAT box

GC box

enhancer

cis-acting element

TATA box (Hogness box)

Cis-acting element

5353

TATA box

5454

RNA-pol does not bind the promoter RNA-pol does not bind the promoter directly. directly.

RNA-pol II associates with six transcRNA-pol II associates with six transcription factors, TFII A - TFII H. ription factors, TFII A - TFII H.

The trans-acting factors areThe trans-acting factors are the prote the proteins that recognize and bind directly oins that recognize and bind directly or indirectly cis-acting elements and rr indirectly cis-acting elements and regulate its activity.egulate its activity.

Transcription factors

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TF for eukaryotic transcription

5656

TBP of TFII D binds TATA TBP of TFII D binds TATA

TFII A and TFII B bind TFII DTFII A and TFII B bind TFII D

TFII F-RNA-pol complex binds TFII BTFII F-RNA-pol complex binds TFII B

TFII F and TFII E open the dsDNA TFII F and TFII E open the dsDNA (helicase and ATPase)(helicase and ATPase)

TFII H: completion of PIC TFII H: completion of PIC

Pre-initiation complex (PIC)

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Pre-initiation complex (PIC)

RNA pol II

TF II F

TBP TAFTATA

DNATF II A

TF II B

TF II E

TF II H

5858

TF II H is of protein kinase activity to TF II H is of protein kinase activity to

phosphorylate CTD of RNA-pol. phosphorylate CTD of RNA-pol. (CTD (CTD is the C-terminal domain of RNA-pol)is the C-terminal domain of RNA-pol)

Only the Only the pp--RNA-pol can move toward RNA-pol can move toward the downstream, starting the the downstream, starting the elongation phase.elongation phase.

Most of the TFs fall off from PIC Most of the TFs fall off from PIC during the elongation phase. during the elongation phase.

Phosphorylation of RNA-pol

5959

The elongation is similar to that of The elongation is similar to that of prokaryotes. prokaryotes.

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

b. Elongation

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RNA-Pol

RNA-Pol

RNA-Pol

nucleosome

moving direction

6161

• The termination sequence is AATAAA followed by GT repeats.

• The termination is closely related to the post-transcriptional modification.

c. Termination

6262

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Post-Transcriptional Post-Transcriptional

ModificationModification

6464

The nascent RNA, also known as The nascent RNA, also known as primary transcript, needs to be primary transcript, needs to be modified to become functional modified to become functional tRNAs, rRNAs, and mRNAs. tRNAs, rRNAs, and mRNAs.

The modification is critical to The modification is critical to eukaryotic systems.eukaryotic systems.

6565

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Primary transcripts of mRNA are called as Primary transcripts of mRNA are called as heteronuclear RNA (hnRNA). heteronuclear RNA (hnRNA).

hnRNA are larger than matured mRNA by hnRNA are larger than matured mRNA by many folds. many folds.

Modification includes Modification includes Capping at the 5Capping at the 5- end - end Tailing at the 3Tailing at the 3- end- end mRNA splicingmRNA splicing RNA edition 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 5a. Capping at the 5- end- end

m7GpppGp----

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ppp5'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

m7Gpppm

2'Npm2'Np

Pi

6969

The 5The 5- cap structure is found on hnR- cap structure is found on hnRNA too. NA too. The capping process occur The capping process occurs in nuclei. s in nuclei.

The cap structure of mRNA will be recThe cap structure of mRNA will be recognized by the cap-binding protein reognized by the cap-binding protein required for translation. quired for translation.

The capping occurs prior to the spliciThe capping occurs prior to the splicing. ng.

7070

b. Poly-A tailing at 3b. Poly-A tailing at 3 - end- end

There is no poly(dT) sequence on the There is no poly(dT) sequence on the DNA template. DNA template. The tailing process d The tailing process dose not depend on the template. ose not depend on the template.

The tailing process occurs prior to thThe tailing process occurs prior to the splicing.e splicing.

The tailing process takes place in the The tailing process takes place in the nuclei. nuclei.

7171

The matured mRNAs are much shorter than the DNA templates.

DNA

mRNA

c. mRNA splicingc. mRNA splicing

7272

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 The structural genes are composed of coding and non-coding regions that arcoding and non-coding regions that are alternatively separated. e alternatively separated.

Split geneSplit gene

EEAA BB CC DD FF GG

7373

Exon and intronExon 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.

7474

mRNA splicingmRNA splicing

7575

Splicing mechanism

7676

lariat

7777

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

intron

pG-OH

pGpA

G pU 3'U5' OH

first transesterification

Twice transesterificationTwice transesterification

second transesterification

U5' pU 3'

pGpA

GOH

5'

3'

7878

Taking place at the transcription Taking place at the transcription levellevel

One gene responsible for more than One gene responsible for more than one proteinsone proteins

Significance: gene sequences, after Significance: gene sequences, after post-transcriptional modification, post-transcriptional modification, can be multiple purpose can be multiple purpose differentiation. differentiation.

d. mRNA editing

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Different pathway of apo B

Human apo B gene

hnRNA (14 500 base)

liverapo B100（ 500 kD） intestine

apo B48（ 240 kD）

CAA to UAAAt 6666

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§3.2 Modification of tRNA

8181

tRNA precursor

RNA-pol III

TGGCNNAGTGC GGTTCGANNCC

DNA

Precursor transcription

8282

RNAase Pendonuclease

Cleavage

ligase

8383

tRNA nucleotidyl transferase

ATP ADP

Addition of CCA-OH

8484

Base modification

（ 1）（ 1）

（ 3）

（ 2）

（ 4）

1. Methylation A→mA, G→mG

2. Reduction U→DHU

3. Transversion U→ψ

4. DeaminationA→I

8585

§3.3 Modification of rRNA

45S transcript in nucleus is the precu45S transcript in nucleus is the precursor of 3 kinds of rRNAs. rsor of 3 kinds of rRNAs.

The matured rRNA will be assembled The matured rRNA will be assembled with ribosomal proteins to form riboswith ribosomal proteins to form ribosomes that are exported to cytosolic somes that are exported to cytosolic space. pace.

8686

rRNA

transcription

splicing

45S-rRNA

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

28S5.8S18S

8787

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

The catalytic RNA is called ribozyme.The catalytic RNA is called ribozyme.

Self-splicing happened often for introSelf-splicing happened often for intron I and intron II. n I and intron II.

§3.4 Ribozyme

8888

• Both the catalytic domain and the substrate locate on the same molecule, and form a hammer-head structure.

• At least 13 nucleotides are conserved.

8989

Hammer-head

9090

Be a supplement to the central dogmBe a supplement to the central dogm

a a

Redefine the enzymologyRedefine the enzymology

Provide a new insights for the origin Provide a new insights for the origin of lifeof life

Be useful in designing the artificial riBe useful in designing the artificial ribozymes as the therapeutical agents bozymes 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