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Transcription
Jacob y Monod
Cytosol
Euchromatin
DNA
mRNA stability
1 Transcription
Nuclear translation
6
Active or inactive protein
1. Specific DNA sequences: Promoters and enhancers 2. Transcription factors: General transcription factors Other transcription factors 3. Chromatin state: Compactation grade of nucleosomes
PRODUCTO
RNA RIBOSOMAL
RNA POL II
Promotores Clase I:
CTCCGAGTCGNNNNNNTGGGCCGCCGG
on
GC
GC
-180
-107
-45
+20
CP (Core Promoter)
G-C rich
UCE y CP son 85% idnticos
C
tRNAs
Extragnicos
DSE
Distal Sequence Element
PSE
TATA
Mixtos
DSE
PSE
TATA
-1.96 kb
-1.56 kb
-200 kb
-100 kb TATA
NF1
NF2 p53
NF4 CRE Oct-2
on TATA
TATA+ INR-
on
TATA
TATA+ INR+
INR
TATA
INR
SL1
RNA POL I
TAF 80 II TAF 60 II TAF 110 TAF 40 II TAF 250 II II TBP TAF 150 II TAF 30 II
TBP
RNA POL II
TFIID
TAF 170 III TAF 70 TBP III
TFIIIB
RNAP I
UBF1
X
1
X 3
SL1
TAF 110 I TAF 48 TBP TAF 63 I I
SL1
TAF 110 I TAF 48 TBP TAF 63 I I
GC
GC
UBF1
UCE
UBF1
CP
RNA 5S
Intragnicos
TFIIIB
tRNA TFIIIB snRNA (7SK, U6)
A B
TFIIIC
Extragnicos
DSE PSE TATA
TFIIIB
TFIIIC
Mixtos
B
DSE
PSE TATA
Histonas
???
NTF-1
RAP74
250
110 150 40
VP16
Sp1
TBP
60
p53
TATA
Inr
DPE
H
EQUIVALENTE A CCG1 CAJA HMG, BROMODOMINIOS UNIN AL PROMOTOR UNIN A SP1 Y A TFIIA
CONSERVACIN EVOLUTIVA Y CARACTERSTICAS DE LAS SUBUNIDADES DEL TFIID DE Saccharomyces cerevisiae, Drosophila melanogaster y Homo sapiens
REPETIDOS WD-40 SIMILITUD A LA HISTONA H4 UNIN A TFIIE Y TFIIF UNIN A MLTIPLES ACTIVADORES
UNIN A CAJA TATA SIMILITUD A LA HISTONA H3 UNIN A p53, VP16 Y TFIIB UNIN AL RECEPTOR DE ESTRGENO
1 / 2 /
X C:G
/ 4 / 5 /
X G:C G:C X
5-T>>c>a=g/A>>t/T>>a=c/A>>t/T>>a/A>>g>c=t/A=T>g>c/G=A>c=t-3
Ciclo de la Transcripcin
Open complex formation
Initiation
Transcript elongation
TATA
Inr
1
A
D
F
Inr
2
F
B
F
Pol II
Inr
Pol II
E H H
PIC
F
E
Terminacin
Pol II
Inr
A
F
P P P P P PP P P P P P P P
Pol II
P P P P
ATP
H
RNA
Terminacin B A
P P P P P PP P P P P P P P
F
P P P P P
B
F
Pol II
Inr
P P P
D
P P P P P P
Pol II
D
P P P P P
PP P P P
TEC
NTPs
Promoter clearence
Checkpoint/Capping
2 5 2 5 2 5
P-TEFb or (Bur1/2 or Ctk1 in yeast)
DR8
Kin28subunit
H
2
5 2 5 2 5 2 5 2 5 2 5
RNAP II
RNAP II
PTase
2 5
Elongation
RNAP II
FCP1 phosphatase
2 5 2
SSu72 phosphatase
3 end processing
RNAP II
Earliest stages of transcription are marked by instability of the transcription complex and a mesaruble tendensy to release the RNA
Stalled RNAP II-DNA complex and short RNA products indicates abortive initiation
Elongation
1. 2. 3.
Active RNAP II
3OH
Pause RNAP II
3OH
Arrest RNAP II
3OH
TFIIS
10.5 Homeodomain from Engrailed protein interacting with its specific DNA recognition site
Kin28subunit
H
2 5 2 5 2 5
2 5 2 5
NELF
DISF
2 5
DISF
RNAP II
RNAP II
RNAP II
5
2 5 2 5 2 5 2 5 2 5 2
P-TEFb
FCP1
2 5 2 5
SPT5
DISF NELF
2 5
DISF NELF
RNAP II
RNAP II
5
CE
PRMT1/5
RNAP II CE
Poly(A)Signal
U rich
AAUAAA conserved
CA
U/GU rich
S. cerevisiae
STOP Eficiency El UAUAUA and related
10-30 nt
Poly(A) site
Py (A)n
E. histolytica
STOP Poly(A)Signal UA(U/A)UU
17-30 nt
Poly(A) site
Formacin y procesamiento del 3 terminal del mRNA en mamferos: corte y poliadenilacin del pre-mRNA
CPSF Cleavage /polyadenilation specificity factor RNA pol II
100 30 73 160
pre-mRNA
3UTR
PAP
CFI
AAUAAA
TAA STOP
77
50
64 Rico en GU
Sitio de corte endonucleoltico
Formacin y procesamiento del 3 terminal del mRNA en mamferos: corte y poliadenilacin del pre-mRNA
CPSF Cleavage /polyadenilation specificity factor RNA pol II
100 30 73 160
pre-mRNA
3UTR
PAPn
CFI
AAUAAA
TAA STOP
Formacin y procesamiento del 3 terminal del mRNA en mamferos: corte y poliadenilacin del pre-mRNA
mRNA
AAUAAA
TAA STOP
100 73
30
160
mRNA FIP1
CPE
CPEBP
(+)
(+)
(+)
Readenilacin citoplsmica
(+)
(-)
5CAP
CBP80 CBP20
Ribonucleasa
POP2
(-)
Factor PAP
Functions PAP has a role in two processes: polyadenylation of mRNA precursors in the nucleus and translational control of certain mRNAs by cytoplasmic elongation of their poly(A) tails. Tissue-specific isoforms (82/77 kDa). Binds RNA, catalyzes the poly(A) synthesis, nonspecific activity by itself, 3-5 exonucleolytic activity, regulated by phosphorylation. Specific activity stimulated by CPSF and PABII. Required for efficient cleavage of 3ends. Interact with CPSF-160 Deadenylase. Component of Ccr4-NOT transcriptional complex
POP2 Deadenylation
Binds poly(A) tail and CPSF-30. Stimulates elongation of poly(A) tail synthesis together PAP. Poly(A) tail length and translation control
Comparison of the cleavage-polyadenylation factors associated with poly(A) signals in mammalians and yeast
Transcription and Polyadenylation factors: Links from the beginning to the end
Components of cleavage-polyadenylation apparatus know to interact with the RNA pol II transcriptional elongation complex in yeast
10.5 Transcriptional activators are modular proteins composed of distinct functional domains
Mammals and plants also DNA methylation -> inherited, maintained during replication.
64
110
290
360
393
Transactivation
Proline-rich
Oligomerization
Basic
Sin3
RNA POL II
SL1(TIF-1B)
RNA POL I
TAF 80 II TAF 60 II TAF 110 TAF 40 II TAF 250 II II TBP TAF 150 II TAF 30 II
TBP
RNA POL II
TFIID
TAF 170 III TAF 70 TBP III
TFIIIB
Promotores Clase I:
CTCCGAGTCGNNNNNNTGGGCCGCCGG
on
GC
GC
-180
-107
-45
+20
CP (Core Promoter)
G-C rich
UCE y CP son 85% idnticos
C
tRNAs
Extragnicos
DSE
Distal Sequence Element
PSE
TATA
Mixtos
DSE
PSE
TATA
-1.96 kb
-1.56 kb
-200 bp
-100 bp TATA
NF1
NF2 p53
NF4 CRE Oct-2
on TATA
TATA+ INR-
on
TATA
TATA+ INR+
INR
TATA
INR
1 / 2 /
X C:G
/ 4 / 5 /
X X G:C G:C
5-T>>c>a=g/A>>t/T>>a=c/A>>t/T>>a/A>>g>c=t/A=T>g>c/G=A>c=t-3
Patikoglou et al.
Histonas
???
NTF-1
RAP74
250
110 150 40
VP16
Sp1
TBP
60
p53
TATA
Inr
DPE
H
EQUIVALENTE A CCG1 CAJA HMG, BROMODOMINIOS UNIN AL PROMOTOR UNIN A SP1 Y A TFIIA
CONSERVACIN EVOLUTIVA Y CARACTERSTICAS DE LAS SUBUNIDADES DEL TFIID DE Saccharomyces cerevisiae, Drosophila melanogaster y Homo sapiens
REPETIDOS WD-40 SIMILITUD A LA HISTONA H4 UNIN A TFIIE Y TFIIF UNIN A MLTIPLES ACTIVADORES
UNIN A CAJA TATA SIMILITUD A LA HISTONA H3 UNIN A p53, VP16 Y TFIIB UNIN AL RECEPTOR DE ESTRGENO
Ciclo de la Transcripcin
Open complex formation
Initiation
Transcript elongation
Not all promoters have all elements Mammals have well-defined start site, but yeast do not
Smale and Kadonaga, Ann. Rev. Biochem (2003), 72:449-79
TAFIIs have two main functions 1. DNA binding 2. Binding other activators They also can have enzymatic activities, like HAT activity
X-links to TAFII250 and TAFII150 When TAFIIs were omitted, no crosslinks were observed TBP doesnt crosslink, presumably because it binds in the minor groove
Footprinting showed that TBP binds, and TAFIIs gave expanded footprint
Fig. 10.10
Essential in yeast
Relieves autoinhibition of TBP Relatively simple: only two subunits
TFIIBN
TFIIF in blue, linkers between domains not resolved at this resolution Conformational changes in RNAP upon TFIIF binding shown in yellow
One subunit of TFIIF (Tgf2 in yeast, Rap30 in human) may be a homolog of bacterial factor
Plays two major roles Phosphorylates the C-terminal domain of RNA polymerase Unwinds DNA at initiation site to create transcription bubble
Model for General Transcription Factors: Initiation, Promoter Clearance, and Elongation
Subsequent initiation events can be more efficient because most of initiation complex is already formed
Model 1: polyA signal leads to changes in composition of RNAP Model 2: mRNA is cleaved at polyA signal, generating new 5-end that is rapidly degraded (torpedo model)
Buratowski, Curr. Opin. Cell Biology (2005) 17, 257-261.
Isolated in 1985 as one of two fractions needed for transcription in vitro from a human rRNA template Composed of three TAFs (TAFI110, TAFI63, and TAFI48) and TBP
Upstream binding factor: UBF in human
Probably only one polypeptide: 97 kDa Binds to upstream promoter element (UPE) to enhance initiation
TATA
Inr
1
A
D
F
Inr
2
F
B
F
Pol II
Inr
Pol II
E H H
PIC
F
E
Terminacin
Pol II
Inr
A
F
P P P P P PP P P P P P P P
Pol II
P P P P
ATP
H
RNA
Terminacin B A
P P P P P PP P P P P P P P
F
P P P P P
B
F
Pol II
Inr
P P P
D
P P P P P P
Pol II
D
P P P P P
PP P P P
TEC
NTPs
Promoter clearence
Checkpoint/Capping
2 5 2 5 2 5
P-TEFb or (Bur1/2 or Ctk1 in yeast)
DR8
Kin28subunit
H
2
5 2 5 2 5 2 5 2 5 2 5
RNAP II
RNAP II
PTase
2 5
Elongation
RNAP II
FCP1 phosphatase
2 5 2
SSu72 phosphatase
3 end processing
RNAP II
Model of the tunable RNA polymerase active site. (a) Presumed mechanism of nucleotide incorporation during RNA polymerization. (b) Presumed mechanism of TFIIS-stimulated hydrolytic RNA cleavage.
Earliest stages of transcription are marked by instability of the transcription complex and a mesaruble tendensy to release the RNA
Stalled RNAP II-DNA complex and short RNA products indicates abortive initiation
X-ray studies of co-crystal of RNAP II and TFIIB show that TFIIB impedes the exit path of the newly formed RNA
Elongation
1. 2. 3.
Active RNAP II
3OH
Pause RNAP II
3OH
Arrest RNAP II
3OH
TFIIS
Human TBP
Ramachandrian plot
Yeast
Land Plants
C. elegans
Drosophila
Vertebrates
10.5 Homeodomain from Engrailed protein interacting with its specific DNA recognition site
Kin28subunit
H
2 5 2 5 2 5
2 5 2 5
NELF
DISF
2 5
DISF
RNAP II
RNAP II
RNAP II
5
2 5 2 5 2 5 2 5 2 5 2
P-TEFb
FCP1
2 5 2 5
SPT5
DISF NELF
2 5
DISF NELF
RNAP II
RNAP II
5
CE
PRMT1/5
RNAP II CE
Poly(A)Signal
U rich
AAUAAA conserved
CA
U/GU rich
S. cerevisiae
STOP Eficiency El UAUAUA and related
10-30 nt
Poly(A) site
Py (A)n
E. histolytica
STOP Poly(A)Signal UA(U/A)UU
17-30 nt
Poly(A) site
Formacin y procesamiento del 3 terminal del mRNA en mamferos: corte y poliadenilacin del pre-mRNA
CPSF Cleavage /polyadenilation specificity factor RNA pol II
100 30 73 160
pre-mRNA
3UTR
PAP
CFI
AAUAAA
TAA STOP
77
50
64 Rico en GU
Sitio de corte endonucleoltico
Formacin y procesamiento del 3 terminal del mRNA en mamferos: corte y poliadenilacin del pre-mRNA
CPSF Cleavage /polyadenilation specificity factor RNA pol II
100 30 73 160
pre-mRNA
3UTR
PAPn
CFI
AAUAAA
TAA STOP
Formacin y procesamiento del 3 terminal del mRNA en mamferos: corte y poliadenilacin del pre-mRNA
mRNA
AAUAAA
TAA STOP
100 73
30
160
mRNA FIP1
CPE
CPEBP
(+)
(+)
(+)
Readenilacin citoplsmica
(+)
(-)
5CAP
CBP80 CBP20
Ribonucleasa
POP2
(-)
Factor PAP
Functions PAP has a role in two processes: polyadenylation of mRNA precursors in the nucleus and translational control of certain mRNAs by cytoplasmic elongation of their poly(A) tails. Tissue-specific isoforms (82/77 kDa). Binds RNA, catalyzes the poly(A) synthesis, nonspecific activity by itself, 3-5 exonucleolytic activity, regulated by phosphorylation. Specific activity stimulated by CPSF and PABII. Required for efficient cleavage of 3ends. Interact with CPSF-160 Deadenylase. Component of Ccr4-NOT transcriptional complex
POP2 Deadenylation
Binds poly(A) tail and CPSF-30. Stimulates elongation of poly(A) tail synthesis together PAP. Poly(A) tail length and translation control
Comparison of the cleavage-polyadenylation factors associated with poly(A) signals in mammalians and yeast
Transcription and Polyadenylation factors: Links from the beginning to the end
Components of cleavage-polyadenylation apparatus know to interact with the RNA pol II transcriptional elongation complex in yeast
10.5 Transcriptional activators are modular proteins composed of distinct functional domains
Figure 9-29
Figure 9-31
9.6 Nonhistone proteins provide a structural scaffold for long chromatin loops
Figure 9-34
10.7 Repressors and activators can direct histone deactylation at specific genes
ubc
2068
Cinasa
HAT
Cinasa
BD BD
TFIID
Ac Ac
Bromodominios
Acetil lisina DNA
Ub
H1 H1 Nucleosoma ?
P P
N
Ub
H1
Ub
Mammals and plants also DNA methylation -> inherited, maintained during replication.
64
110
290
360
393
Transactivation
Proline-rich
Oligomerization
Basic
Sin3