Transcription---- Biosynthesis of RNA Chapter 13.
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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
Transcription----Biosynthesis of RNA
The template and enzyme
The transcription in prokaryotes
The transcription in eukaryotes
The processing of post-transcription
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.
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
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.
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:
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
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
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).
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?
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