Transcription---- Biosynthesis of RNA Chapter 13.

Transcription----

Biosynthesis of RNA

Chapter 13

Transcription----Biosynthesis of RNA

Introduction

1. Transcription is the first stage of the process of gene expression.

2. Transcription processes have to suffer strictly regulation to meet the need of development, morphogenesis and physiological functions of organisms .

3. The products of transcription are RNA

Transcription

RNADNA

The process which RNA polymerase catalyzes the yield of RNA (tRNA, mRNA, rRNA ) with one of double strands of DNA as template, NTPs as precursors, in the light of the rule of complementary base pairing.

Precursors : NTP (ATP, UTP, GTP, CTP)

Template : DNA

Enzyme: RNA polymerase, RNA-pol

Other proteins

The requirements of transcription

Replication Transcription Template both strands of DNA one of DNA strands

Precursors dNTP NTP

Base pairing A→T, G→C A→U, T→A, G→C

Polymerase DNA polymerase RNA polymerase

Product DNA tRNA, mRNA, rRNA

Primer

Comparison of replication and transcription

General picture of transcription

Transcription----Biosynthesis of RNA

The template and enzyme

The transcription in prokaryotes

The transcription in eukaryotes

The processing of post-transcription

Section 1

The template and

enzyme

1. The template :

For one gene, only one of DNA strands can serve as template.

So, there are template strand ( Watson strand ) and coding strand ( Crick strand ).

The most important character of transcription is asymmetric transcription.

Structural gene:

DNA Transcription plot

5’……GCAGTACATGTC…………3’3’……c g t c a t g t a c a g…………5’ DNA

5’……GCA GUA CAU GUC………3’ mRNA

N…... Ala Val His Val.. ………….Cpeptide

transcription

translation

Note ： capital letters means the code strand, small letters means the template strand

Asymmetry transcription

Structure gene

Template strand

5’3’ 5’

3’

Arrowhead means the direction of transcription

5’ 5’

5’

Coding strand

For asymmetric transcription, there are two meanings:

1) Only one strand of a gene can serve as

template, the other which is complimentary to

the template strand can’t be transcripted.

2) Not all the template strands of genes are

found in the same strand on DNA molecule.

2. RNA polymerase

The enzyme related to transcription i

s RNA polymerase, which is termed as D

NA dependent RNA polymerase (DDRP)

or RNA pol, or transcriptase.

only one RNA pol has been found:

α2ββ’ σ (holoenzyme )

α2ββ’ ( core enzyme )

Core enzymeHoloenzyme

1) RNA polymerase in prokaryotes

Holoenzyme: It can initiate transcription specifically at promoter sites and catalyze polymerization of two free NTPs.

Core enzyme: It can not initiate transcription specifically at promoter sites but can catalyze RNA elongation.

Core enzyme: the RNA polymerase without the subunit is called core enzyme (2).

There are more than one kind of factor in prokaryotes

70 carries out the promoter recognition process on their own, mainly responsible for the housekeeping gene expression.

32 is responsible for the heat-shock gene expression under some emergent cases.

‘Housekeeping’ genes are those that encode many proteins needed for routine cell functions and which are therefor expressed at low rates in all cells.

RNA polymerase binds to DNA at the Transcriptional startpoint

There are three kinds of RNA pol:

RNA pol I : 45S rRNA

RNA pol II : hnRNA

RNA pol III : tRNA, 5s rRNA, snRNA

2) RNA polymerase in Eukaryotes

RNA synthesis in Prokaryotes

Three phases of transcription:

Initiation of transcription

Elongation of transcription

Termination of transcription

Section 2

During initiation, RNA polymerase recognizes promoter site, and then unwinds DNA locally to expose a single-stranded DNA template that can be transcripted.

1. Initiation of transcription

The transcriptional unit in prokaryotes is operon that consists of two regions on DNA:

Regulation region

Structural gene region

Regulation region Structural gene region

Operator gene

Promotor, the region for binding of RNA pol

Inhibitor gene

P OI

-50 -40 -30 -20 -10 1 10

TTGACA TATAATPu

pppG

NH2

Operon structure

1 2

Pribnow box

Inhibitive protein

substrate

mRNA

The steps of transcription initiation

1) The sigma factor in holoenzyme of RNA pol recognizes the promotor and let the whole enzyme to bind with the promotor sequence.

2) To unwind the local region of promotor on DNA.

3) To form a initiation complex of transcription.

Holoenzyme- DNA-pppGpN-OH

TTGACA TATAAT-35-10

Transcriptional start site

3`

+1

pppGpN-OH

Transcriptional bubble

RNA pol

pppG-OH + pppN-OH pppGpN-OH + PPi

Holoenzyme of RNA pol

-10 sequence: Located about 10 nucleotides upstream of where transcription will begin.-35 sequence: Located about 35 nucleotides upstream.

TTGACA TATAAT-35 -10

Transcriptional start site

5`

By convention, the first nucleotide of the template DNA that is transcribed into RNA is denoted +1, the transcriptional start site.

+1

Two important sequence in prokaryotic promoters:

2. Elongation of transcription

The elongation phase of RNA synthesis, which begins after formation of the first bond, is therefore carried out by core enzyme.

The transcriptional complex:

Core enzyme of RNA pol-DNA template-new RNA

The Core enzyme(2) moves along the gene, synthesizes a complementary RNA copy to the DNA template, using four ribonucleoside 5` triphosphates (ATP,CTP,GTP,UTP) as precursors.

The 3`-OH at the end of the growing RNA chain attacks the phosphate groups of the incoming ribonucleoside 5` triphosphate to form a 3`5` phosphodiester bond.

-subunit dissociates from the enzyme, once transcription has been initiated .

The complex of RNA polymerase, DNA template and new RNA transcript is called a transcription bubble. Because within it there is a region where the DNA double helix has opened up to allow transcription to occur.

The RNA transcript forms a transient RNA-DNA hybrid helix with its template strand but then peels away from the DNA as transcription proceeds.

Transcription bubble

Direction :

RNA is synthesized in the 5`3`direction. DNA template in the 3` 5` direction.

The DNA is unwound ahead of the transcription bubble, and after the transcription complex has passed , the DNA rewind.

DNA template strand

DNA template strand

OH

OH

OH

OH

U U

Transcription by RNA polymerase. In each step the incoming ribonucleotide selected is that which can base-pair with the next base of the DNA template strand. In the diagram, the incoming nucleotide is rUTP to base-pair with the A residue of the template DNA, A 3`5`phosphodiester bond is formed, extending the RNA chain by one nucleotide, and pyrophosphate is released. Overall the RNA molecule grows in a 5`3`direction.

direction

5`

3`

Termination of transcription in prokaryotes

Transcription continues until a termination signal is reached. There are two forms in the termination:

2) A mechanism of dependent on rho () factor

1) A mechanism of not-dependent on rho () factor

The simplest termination signal is a GC-rich region in the template that is a palindrome, followed by an AT-rich sequence. The RNA made from the DNA palindrome is self-complementary and so base-pairs internally to form a hairpin structure followed by a few U residues.

1 ） A mechanism of not-dependent on rho () factor

GC

UU G

A-U-U-U-U-OH 3`

G•CA•UC•GC•GG•CC•GC•GG•C

5`

3`---------CGGCGGTCAAGCCGACCGCCG-------TAAAA-OH 5`

5`---------GCCGCCAGUUCGGCUGGCGGC-------AUUUU-OH 3`

Transcription

A typical hairpin structure formed by the 3` end of an RNA molecule during termination of transcription.

5`---------GCCGCCAGTTCGGCTGGCGGC-------ATTTT-OH 3`

template

RNA

A mechanism of not-dependent on rho () factor

Those that lack such a structure require an additional protein, called rho (), to allow recognition of the termination site and stop transcription.

2) A mechanism of dependent on rho () factor

Rho (), can bind to the 3’ end of RNA and to change the conformation of RNA pol, therefore to loose the binding RNA pol with DNA template, then release out from the bubble.

5`5`

Promoter Structure gene Termination

Gppp

5`pppG

5`pppG

5`mRNA

Core enzyme

holoenzyme1

2

3

4

5

6

7

8

1,2. The gene waiting transcription, the single strand with 3`5` is template strand.

3,4. Initiation ,holoenzyme binds to promoter . 5.the first pppG is added.

6. After dissociates from the enzyme, the elongation begins.

7. Termination, factor is added, core enzyme releases;

8. Finish of the transcription.

53

DNA

Ribosome

RNA

RNA pol

RNA post-transcriptional modification

In prokaryotes, mRNA requires little or no modification prior to translation.

rRNA:

(1) Cleavage: the rRNA precursor molecule is cleaved by specific ribonucleases to yield mature 23s,16s and 5s rRNA.

(2) Methylation of some bases and ribose moieties of rRNA also occurs.

mRNA:

16S rRNA tRNA 23S rRNA 5S rRNA

An RNA precursor molecule that is cleaved to yield the 23S, 16S and 5S rRNA and a tRNA molecule; the spacer RNA( open blocks) is degraded during these processing steps.

(1) Cleavage: the tRNA precursor molecule is cleaved by specific ribonucleases .

(2) Some tRNA molecules further require the addition of the three nucleotides CCA to the 3` end before they can function.

tRNA:

RNA synthesis in Eukaryotes

Section 3

Gene transcription in eukaryotes

The transcription in eukaryotes and prokaryotes is similar, but also there are some difference.

overview

1) RNA polymerase, there are three kinds of RNA pol in eukaryotes, but only one kind in prokaryotes.

2) The process of transcription are different during the initiation and the termination.

RNA polymerases—pol I, pol II, pol III

RNA polymerase I Located in the nucleolus and transcribes the 28s, 18s and 5.8s rRNA genes.RNA polymerase II Located in the nucleoplasm and transcribes mRNA from protein-coding gene as well as most small nuclear RNA(snRNAs) involved in mRNA processing .

In eukaryote cells, there are three kinds of RNA polymerases.

Located in the nucleoplasm and transcribes the genes for tRNA, 5s rRNA, snRNA, and the 7s RNA associated with the signal recognition particle (SRP) involved in the translocation of proteins across the endoplasmic reticulum membrane.

RNA polymerase III

RNA synthesis in eukaryotes

It is similar to the process in prokaryotes.

Initiation: RNA polymerase combines with the promoter site upstream of the transcriptional start site.

Template: only one strand of a double helix DNA acts as a template.

Primer: does not require a primer.

Direction: The synthesis direction is 5`→3`.

Transcription of mRNA genes

mRNA gene organization

Exons

The sequence of protein-coding sections in a gene.

Introns

The sequence of nonprotein-coding sections in a gene.

Split gene

The vast majority of protein-coding genes in eukaryotes are discontinuous. The exons are interrupted by introns.

The triplet codons within the exons and the order of exons in the gene is consistent with the amino acid sequence of the encoded polypeptide.

CA B D

coding region A 、 B 、 C、 D

noncoding region

AATAA

(A point for cutting and adding a tail )

Cis-acting elements in eukaryotes

-25bp-70~-80bp

-110bp

enhancer

Promoter Structural gene

Initiation site

exon intron

Modification site

Real termination site

GC CAAT TATA

5`cap added 3`poly(A) added

promoter termination region

Exon 1 Exon 2 Exon 3

Intron1 Intron2

5`pppPrimary RNA transcript

Cleavage by endonuclease and addition of poly(a) tail

5` AAAA200 3`

poly(a) tailcapRNA slicing

1 2 3

Transport to cytoplasm via nuclear pore

Structure and expression of a protein-coding gene in

eukaryotes

5` AAAA200 3`

Initiation of mRNA transcription

The enzyme---- RNA polymerase II

Promoter

Most promoter site include a sequence located about 25 bp upstream (i.e. to the 5` side) of the start site which has the consensus TATAAA and is called the TATA box.

TATA box sequence resembles the -10 sequence in prokaryotes(TATAAT) except that it is located further upstream.

General transcription factors They are proteins or protein complex that RNA polymerase II requires to assemble into a complex on the promoter in order for RNA polymerase to bind and start transcription. These all have the generic name of TFII (for transcription factor for RNA polymeraseII).

Trans-acting factors :

Transcriptional factors and their functions

TF Mw （ KD) Functions

TF II ATF II BTF II DTF II E

TF II F

12, 19, 35333834(β),57(α)

30, 74

To stable the binding of TF II DTo improve the binding of pol II

To recognize TATA box

ATPase

Helicase

The forming of transcription initiation complex

(1) TFIID binds to the TATA box. The key subunit of TF II D is TBP(TATA box-binding protein).(2) TFII A binds, followed by TF II B.(3) RNA polymerase II, which has already complex with TFIIF,binds followed by the binding of TFIIE,H and J.(4) The transcription begins when forming of transcription initiation complex. (5) It is a basal transcription apparatus. Transcription is only at a low rate. For a high rate of transcription, other transcription factors are required.

TFIID

D

TFIIA, TFIIB, TFIIF, RNA polymerase II

DA B

TFIIE, TFIIH, TFIIJ

DA B

Transcription starts

Initiation of transcription by RNA polymerase II. TFIID binds to the TATA box followed in order by the binding of TFIIA,TFIIB and a pre-formed complex of TFIIF-RNA polymerase II. Subsequently TFIIE, TFIIH and TFIIJ bind in order and transcription then starts about 25 bp downstream from the TATA box.

Note that the placement of the the various factors in this diagram is arbitrary; their exact position in the complex are not yet known.

TATA

TATA

Pol.II

F

Pol.II

F E H J

Elongation and termination of mRNA transcription

Elongation When the transcription initiation complex is formed, the transcription begins and the elongation of the RNA chain continues until termination occurs.Termination Unlike RNA polymerase in prokaryotes, RNA polymerase II does not terminate transcription at specific sites but rather transcription stops at varying distances downstream of the gene.

TTATTT

AAUAAA

3’5’

AATAAA

5’ AAA········AAA

Termination and post-modification of mRNA transcription

Modification site

Adding of a poly A tail

RNA pol II

The site of termination

mRNA processing of post-transcription

The primary transcript of mRNA is immature, it needs processing in order to create mature mRNA ready for translation; this involves capping, polyadenylation and RNA splicing.

Capping: 5’ end with m7Gppp-GpN

Polyadenylation: a poly A tail on 3’-end

RNA splicing: to cut out the introns, and join the exons

RNA editing: such as apolipoprotein B mRNA

Capping of mRNA

The structure of capping The 5` end of the primary transcript is modified by the addition of a methylated guanine Cap(m7Gppp).

The structure of capping of mRNA

5’-pppG……Phosphatase

Pi

5’-ppG… 5’-GpppG…pppG ppi

Methylation

CH3

mGpppG…

GO

OH OH

CH2-O-P-O-P-O-P-O

H2C O

O OH

PO

O-

O …AAA

G

O O O

O- O- O-

The form of 5’-end cap

1’

5’

5’

1’3’

3’

The roles of capping

A. Protecting mRNA from degration by ri

bonucleases that have specificity for 3`5`phosph

odiester bonds and cannot hydrolyze the 5`5` b

ond in the cap structure.

B. The cap plays a role in the initiation ste

p of protein synthesis in eukaryotes.

Polyadenylation

The endonuclease cleaves the RNA transc

ript at a site approximately 10-30 nucleotides

on the 3` side of a AAUAAA sequence that is c

alled a polyadenylation signal. Poly(A) polyme

rase then adds 100-200 adenosine monophosp

hate residues to the new 3` end using ATP as

precursor.

The roles of polyadenylation

To protect the 3` end of the final mRNA against ribonuclease digestion and hence stabilizes the mRNA.

To increase the efficiency of translation of the mRNA.

RNA splicing

To cut out intron precisely.

To join the neighboring exons to produce a functional mRNA molecule.

A lariat intermediate is formed first, to let the two neighboring exons near together, the intron become a loop round.

A snRNP is needed which consists of snRNA and some proteins, called a spliceosome .

snRNAs-----small nuclear RNAs, each of which is associated with several proteins to form a small nuclear ribonucleoprotein particle or snRNP.

The roles of snRNAs: To align the splice site ready for splicing.As a catalyst, to catalyze the splicing

Exon 1 Exon 2Intron

②

③

UACUACA - AG

UG

U4

U5

U6

E1

E2

U1 U2

UACUACA - AG

UGU6

E1

E2

U1 、 U4 、 U5

U2

pG-OH(ppG-OH, pppG-OH)

U-OH

GpUpGpA

UpA GpUExon 1 Intron Exon 1

G-OH

UpU

pGpA

Twice transesterification

The first transesterification

The first transesterification

gene

hnRNA

adding cap and tail

hnRNA splicing

matured mRNA

RNA editing on an mRNA molecule

RNA editing

The nucleotide sequence on an mRNA molecule may be changed by several reactions other than RNA splicing.

Individual nucleotides within the mRNA m

ay be changed to other nucleotides, deleted enti

rely or additional nucleotides inserted. The effe

ct of RNA editing is to change the coding capaci

ty of the mRNA so that it encodes a different po

lypeptide from that originally encoded by the g

ene. Such as apo B mRNA

apo B mRNA ( liver)

apo B100

apo B mRNA ( small intestine)

apo B gene

apo B48

6666 C→U CAA→UAA(glutamine)

(Mw 500 000) (Mw 240 000)

Post-transcriptional modification of rRNA

The gene of rRNA, or rDNA, belongs to abundant gene. There are more than 100 copies of rDNA on the same DNA molecule.

Similarly, the genes for tRNA, histone, 5S rRNA clustered on DNA too.

Organization and expression of rRNA genes

45S rRNA precursor

18S - rRNA 5.8S 和 28S-rRNA

rDNA

内含子 内含子 28S5.8S18S

Transcription

Cleavage

rRNA genesUntranscribed spacer

tRNA molecules from all organisms are base-paired internally to give a ‘clover-leaf’ structure( secondary structure). This consists of three stem-loop. Each tRNA has a 5`-phosphate group and the nucleotide sequence CCA at its 3` end with a 3`-OH group.

The cloverleaf structure folds further into an L-shaped conformation(tertiary structure).

Post-transcriptional modification of tRNA

Post-transcriptional modification of tRNA

1) To cut the intron sequences such as the 5’ end and 3’ end excess sequences, the excess sequence on the anticodon ring.

2) To add the 3’ end CCA-OH.

3) To modify some bases on the special sites to form some special scarce bases, such as DHU, Am, I ( inosine ), ψ(psi).

Transcriptional modification of tRNA

The structure of the simplest ribozyme

Substrate site

Questions

1. Describe the process of transcription in prokaryotes, how does it be terminated?

2. Compare RNA polymerases in prokaryotes with those in eukaryotes

3. What are the characters of RNA transcription?

4. Compare transcription with replication from template, enzyme, precursors, base-paring, products, characters

5. How do the primary transcripts of mRNA genes be modified in eukaryotes?

6. How do the primary transcripts of tRNA genes be modified in eukaryotes?

7. What is asymmetric transcription?