transcriptiontranslation Reverse transcription " The Central Dogma of molecular biology"...

transcription translation

Reverse transcription

" The Central Dogma of molecular biology"

replication

Chapter 10

Transcription (RNA Biosynthesis)

RNADNA

•products：mRNA tRNA rRNA

Transcription*:

RNA biosynthesis from a DNA template

is called transcription.transcription

Enzymes and Proteins involved in transcription ：

•substrates : NTP

( ATP, UTP, GTP, CTP )

•template: DNA

• enzyme : RNA polymerase

• the other Protein factors

Chemical reaction-- polymerization reaction: RNA polymerase catalyze formation of Phosphodiester bonds and release pyrophosphate (ppi)

RNA precursor

RNA polymerase

RNA biosynthesis is similar to DNA biosynthesis*:

Template- DNA

Enzyme—dependent on DNA

Chemical reaction--the formation of Phosphodiester bonds

Direction of synthesis--- 5’ 3’

obey the ruler of base paired

RNA biosynthesis includes three stages: Initiation: RNA polymerase binds to the

promoter of DNA, and then a transcription “bubble” is formed.

Elongation: the polymerase catalyzes formation of 3’5’-phosphodiester bonds in 5’3’ direction, using NTP as building units.

Termination: when the polymerase reaches a termination sequence on DNA, the reaction stops and the newly synthesized RNA is released.

Formation of a transcription bubble

1. RNA biosynthesis in prokaryotes

RNA polymerase in E. coli :

consists of five subunits, 2’, which is called

“holoenzyme”. The subunit functions as a starting

factor that can recognize and bind to the promoter

site.

The rest of the enzyme, 2’, is known as “core

enzyme”, responsible for elongation of the RNA

sequence.

E. coli RNA polymerase

Core enzyme Holoenzyme

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35RNA-pol

1) Important terms in RNA biosynthesis.

A)Operon*: a coordinated unit of gene expression, which usually contains a regulator gene and a set of structural genes.B) Promoter site*: a region of DNA templates that specifically binds RNA polymerase and determines where transcription begins.

regulator gene structural genesPromoter site

The –10 sequence: refers to the consensus TATAAT, and is known as “Pribnow box”.

The –35 sequence: refers to the consensus TTGACA, which is recognized by the subunit of RNA polymerase,

DNA template TTGACA TATAAT

-35 +1-10

Pribnow boxRNArecognition site

consensus

sequences

T T G A C A

A A C T G T

-35

(Pribnow box)

T A T A A T Pu A T A T T A Py

-10

1-30-5 0 10-10-40 -205

3

3

5

recognition site

regionregion

the site of transcription (the start site)

C) Sense and antisense strand: The antisense (-) strand refers to the DNA strand

that is used as template to synthesize mRNA.

The sense (+) strand of a DNA double helix is the

non-template strand that has the same sequence as that

of the RNA transcript except for T in place of U.

Antisense (-) strand = template strand

Sense (+) strand = coding strand

5

3

3

5

coding strand

structural gene

template strand

antisense strand

sense strand

Sense and antisense strand:

3) Process of RNA biosynthesis: The process is similar to DNA synthesis but no primer is needed and T is replaced by U.

A) Initiation:•σ factor recognizes the initiation site （ -35 region ） , the

holoenzyme of RNA-pol bind to duplex DNA and move

along the double helix towards –10 region.

•the holoenzyme of RNA-pol arrived on –10 region ， and

bind to –10 region ， DNA is partially unwound and was

opened 10-20 bp length.

•Then incoming 2 neighbour nucleotides which base pairs are complementary with DNA template, RNA polymerase catalyzed the first polymerization reaction.

– 5’ -pppGpN – OH + ppi

RNApol(α2ββˊσ)-DNA-pppGpN-OH3’

pppG NTPpppGpN - OH

ppiinitiation complex:

5’-pppG -OH + NTP

DNA template TTGACA TATAAT

+

“Core”

DNA template TTGACA TATAAT

DNA template TTGACA TATAAT

DNA template TTGACA TATAAT

The first phosphodiester bond formed

B) Elongation: after the first phosphodiester bond has been formed, the subunit is released. The core enzyme moves in a 5’3’ direction on the DNA strand while it is catalyzing elongation of the RNA transcript.

DNA template TTGACA TATAAT

DNA template TTGACA TATAAT

DNA template TTGACA TATAAT

NTP

RNA-pol (core enzyme) ···· DNA ···· RNA

tanscription complex:

Sense strandRNA polymerase

Antisense strand

Newly synthesizedRNA strand

5’PPP

Rewinding

Unwinding

Direction oftranscription

5’3’

3’5’

C) Termination: when the core enzyme reaches a termination sequence, the region near the 3’end of RNA forms a hairpin structure by self base-pairing. The transcription stops, the core enzyme and the newly synthesized RNA are released.

For those DNA templates that lack the sequence to produce a hairpin structure of the RNA transcript, a protein factor called “” recognizes the termination site, stops transcription, and causes release of the newly synthesized RNA.

A hairpin structure at the 3’end of RNA

CU GU G

G • CA • UC • GC • GG • CC • GC • GG • C

A-U-U-U-U-OH 3’5’

Termination by hairpin structure of RNA

5’pppG

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35

RNA-polymerase

53

DNADNA

The multiple-site transcription in bacteria

RibosomeRibosome

RNARNA

RNA polymeraseRNA polymerase

Subunits of RNA polymerase in E. coliSubunit Size (AA) Function 329 required for assembly of the enzyme; interacts with some regulatory proteins; involved in catalysis 1342 involved in catalysis: chain initiation and elongation' 1407 binds to the DNA template 613 directs the enzyme to the promoter 91 required to restore denatured RNA polymerase in vitro to its fully functional form

4) Post-transcriptional modification:

The newly synthesized precursors of rRNA and tRNA in bacteria undergo a series of process.

A) Processing of rRNA: the 16S, 23S, and 5S rRNAs in prokaryotes are produced by cleavage of a rRNA precursor, catalyzed by ribonuclease III. Additional processes include methylation of bases and sugar moieties of some nucleotides.

Processing of rRNAs

16S 23S 5S

16S 23S 5S

16S(1.5kb) 23S(2.9kb) 5S(0.12kb)

First cleavage

Second cleavage

B) Processing of tRNA: The removal of the 5’ end of tRNA precurs

ors is catalyzed by RNase P. RNase P is a ribozyme consisting of RNA that possesses enzyme activity.

Other processes include the addition of nucleotides (CCA) to the 3’-end of tRNA, and formation of some unusual residues such as pseudo-U, I, T, methyl-G, and DHU, etc.

Modification of some residues in tRN

As

4) Inhibition of transcription: Rifampicin: an antibiotic that specifically in

hibits the initiation of transcription by blocking the formation of the first several phosphodiester bonds in RNA biosynthesis.

Streptolydigin: binds to bacterial RNA polymerase and inhibits elongation of RNA chain.

Actinomycin D: binds to DNA and prevents transcription (at low concentrations it doesn't affect DNA replication)

2. RNA biosynthesis in eukaryotes

1) RNA polymerases in eukaryotes: three enzymes, each of which contains 12 or more subunits.

Polymerase location RNAs transcribedPol I nucleolus 28S, 18S, 5.8S rRN

APol II nucleoplasm pre-mRNA, snRNAPol III nucleoplasm tRNA, 5S rRNA, U6 snRNA, 7S RNA

2) Process of eukaryotic RNA synthesis

A) Initiation: similar to Pribnow box, a start site consensus (called TATA box) at –25 is required for the recognition by RNA polymerase in eukaryotes.

A ATATA A

T TStructural gene

+1-25

Pol II requires several transcription factors to start transcription:

TFII-A: to stabilize the TFIID-TATA box complex;

TFII-B: to link Pol II to the initiation complex;

TFII-D: to recognize and bind to the TATA box;

TFII-E: to interact with Pol II and TFII-B;

TFII-F: to form Pol II-TFIIF complex. It also has

DNA helicase activity;

TFII-H, -J: to form the initiation complex.

TA TA box Structural gene

TBPSTFII-D

TBPS

BA

TBPSA B

Pol II F

TBPSA B

F

JHE

TBPSA B

F HJ

E

B) Elongation : after the initiation complex has formed, the RNA polymerase catalyzes transcription in a 5’3’direction, using the (-) DNA strand as template.

Soon after the 5’end of the extending RNA chain appears from the polymerase complex, a cap structure is added at the end.

+

O

H

OCH3

H

O

H

O

H

OCH3

H

O

H

O

H

OH

H

OH

CH2

H

OP

O-O

O

P

O

O-O

P

O

-O O

HN

N N

N

O

H2N

CH3

CH2 Base

H

H

P-O O

O

CH2 Base

H

Cap structure of mRNA

7-methylguanylate

C) Termination: Two mechanisms may cause termination of RNA transcription:

A hairpin structure formed at the 3’end of the nascent RNA causes stop of transcription, as is seen in the prokaryotic RNA synthesis.

A stop signal sequence, AAUAAA, near the 3’end results in the recognition and binding by a specific endonuclease, which cleaves the nascent RNA chain and stops transcription. The newly synthesized mRNA precursor is then added a poly A tail by poly A polymerase.

Cleavage and polyadenylation of a mRNA precursor

AAUAAA

Template DNA

Nascent RNA Cleavage signal

AAUAAA AAAA(A)n-OH 3’5’

endonuclease

ATP

PPi

Poly A polymerase

RNA polymerase

mRNA precursor

3) Processing of eukaryotic RNA precursors:

A) Gene organization: protein-coding genes in eukaryotic DNA are organized in a discontinuous fashion. The protein-coding sections are called “exons”, which are interrupted by noncoding sections called “introns”.

Promoter

Transcriptioninitiation site

Exon 1 Exon 1Exon 1

Intron 1 Intron 2

TranscriptionTermination region

B) RNA splicing: a process in which introns of a pre-mRNA are removed to produce a functional mRNA.

Promoter

Exon 1 Exon 3Exon 2

Intron 1 Intron 2Promoter

Exon 1 Exon 3Exon 2

Intron 1 Intron 2

Exon 1 Exon 3Exon 2

Intron 1 Intron 2

5’ AA(A)250 3’Exon 1 Exon 3Exon 2

Intron 1 Intron 2

5’ AA(A)250 3’

5’ AA(A)250 3’5’ AA(A)250 3’

Transcription

RNA splicing

C) Steps in RNA splicing: usually the exon-intron boundaries are marked by specific sequences. The intron starts with GU and ends with AG.

Intron

Exon 1 Exon 2GU AGU/C11CURAYExon 1 Exon 2GU AGU/C11CURAY

5’ splice site 3’ splice site

Branch pointsequence

Polypyrimidinetract

I. Formation of a lariat intermediate: the phosphodiester bond of the 5’ splice site is attacked by the 2’-OH of the residue A in the branch point, forming a 2’5’bond and releasing the exon 1 with a new 3’-OH end.

II. Connection of exons: The new 3’-OH end attacks the phosphodiester bond at the 3’splice site causing the two exons to join and releasing the intron.

RNA splicing requires the small nuclear ribonucleoprotein particles (snRNP), each of which consists of a small nuclear RNA and several proteins. They are named U1, U2, U3….

snRNPs bind to the pre-mRNA to form a complex, called spliceosome, which brings the two neighbored exons together for splicing.

E x o n 1 E x o n 2G U A GA

U 1 U 2

E x o n 1 E x o n 2G U A GA

U 1 U 2

s n R N P s

E x o n 1 E x o n 2G U A GA

U 1

U 2U 6

U 5U 4

U 4 - U 5 - U 6

Spliceosome

Exon 1 Exon 2GU AGA

U1

U2U6

U5U4

Exon 1 Exon 2GU AGA

U1

U2U6

U5U4

GU AG-OH 3’A

U1

U2U6

U5U4Exon 1 Exon 2

Lariat intermediate

4) Alternative processing:

A) Alternative polyadenylation sites: this will cause different splice-sites and produce different mRNAs with varied lifetimes.

Exon 1 Exon 3Exon 2

Poly A Poly A

Splicing

B) Alternative splicing: will cause different combinations of exons from a primary transcript of a single gene. This may be resulted from regulatory proteins that control the use of certain splice-sites.

Exon 1 Exon 3Exon 2

Splicing

5) RNA editing: refers to the reactions that can change the nucleotide sequence of an mRNA molecule by non-splicing mechanisms. The change may include: nucleotide(s) change, deletion, and insertion.

e.g. the mRNA for apolipoprotein B in the liver is translated to apolipoprotein B100, while in the small intestine the mRNA is changed to yield a new termination codon (UAA), resulting in a much shorter protein, apolipoprotein B48.

CAA

UAA

Apolipoprotein B mRNA

Trans

latio

nNH4

Editing (deamination)

translation

Lipoproteinassembly

Lipoproteinassembly

LDL receptorbinding

ApoB100

ApoB48

Edited mRNA

3. Reverse transcription and RNA replication

1) Reverse transcription: biosynthesis of DNA using RNA as a template.

It is important for some viral infections. These viruses are called retroviruses, such as some tumor viruses and HIV.

Reverse transcription is also a powerful tool in molecular biological techniques or genetic engineering, such as RT-PCR.

2) RNA replication: RNA replication occurs in some viruses.

These viruses encode RNA-directed RNA polymerase that catalyzes biosynthesis of RNA from an RNA template.

RNA replication helps the RNA viruses easily reproduce their progeny viruses.