CENTRO DE INVESTIGACIN Y DE ESTUDIOS AVANZADOS DEL INSTITUTO POLITCNICO NACIONAL

Transcription

Dr. Juan Pedro Luna Arias Departamento de Biologa Celular

Opern de la lactosa: Un modelo de represin

Jacob y Monod

Eukaryotic Control of Gene Expression

Nucleus
Heterochromatin Inactive mRNA siRNA interference

Cytosol

Euchromatin
DNA

mRNA maturation pre-mRNA mRNA

mRNA stability

mRNA Translation 4 mRNA transport Protein

Post-translational modifications

1 Transcription
Nuclear translation

6
Active or inactive protein

Whats the transcription process of genetic information transfer?

What are the key players in Gene Expression?

1. Specific DNA sequences: Promoters and enhancers 2. Transcription factors: General transcription factors Other transcription factors 3. Chromatin state: Compactation grade of nucleosomes

RNA POLIMERASAS EUCARITICAS

ENZIMA
RNA POL I

PRODUCTO
RNA RIBOSOMAL

RNA POL II

RNA MENSAJERO (mRNA) RNAs NUCLEARES PEQUEOS

RNA POL III

RNA DE TRANSFERENCIA (tRNA) RNA VIRALES Y CELULARES RNA RIBOSOMAL 5S

Promotores Clase I:
CTCCGAGTCGNNNNNNTGGGCCGCCGG

on

GC

GC

-180

-107

-45

+20

UCE (Upstream Control Element)

CP (Core Promoter)

G-C rich
UCE y CP son 85% idnticos

Promotores Clase III:

Intragnicos
+55 +80
RNA 5S X. laevis

C
tRNAs

Extragnicos
DSE
Distal Sequence Element

PSE

TATA

Proximal Sequence Element

Mixtos

DSE

PSE

TATA

Estructura de un promotor de tipo II eucarintico

Binding sites for activators that control transcription of a hypothetical gene
Enhancer Promoter-proximal region Core Promoter

-1.96 kb

-1.56 kb

-200 kb

-100 kb TATA

NF1

NF2 p53
NF4 CRE Oct-2

GR NF8 RAR NF10

YY1 C/EBP AP-1

Estructura bsica del ncleo del promotor (100 pb)

on TATA

TATA+ INR-

on
TATA

TATA+ INR+

INR

on TATA- INR+ INR on TATA- INR-

Other Elements of the Core Promoter

TATA

INR

TFIIB recognition element DPE (Downstream Promoter Elements)

TAF 110 I TAF 48 TBP TAF 63 I I

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

RNA POL III

TFIID
TAF 170 III TAF 70 TBP III

TFIIIB

Transcripcin por RNA Pol I

SL1
TAF 110 I TAF 48 TBP TAF 63 I I

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

Transcripcin por RNA pol III

TFIIIA TFIIIC

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

SIMILITUD A LA HISTONA H2B

TATA BOX DEFINITION

5-TATAAAAG-3

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

TBP induce una dramtica distorsin en el DNA

Ciclo de la Transcripcin
Open complex formation

Pre-initiation complex assembly

Initiation

Termination Promoter clearence

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

YSPTSPS: 26 yeast 32 C. elegans 45 in Drosophila 52 in mammals

FCP1 phosphatase

2 5 2

SSu72 phosphatase
3 end processing

RNAP II

The phosphorylation cycle of the CTD of the large subunit of 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

Newly initiated RNAP II has a tendency to slip laterally

Slip of RNAP II is greatly reduced after RNA-DNA hybrid is 8-9 nt in length

Slip of RNAP II is undetected after RNA is 23 nt long

X-ray studies of co-crystal of RNAP II and TFIIB show that TFIIB impedes the exit path of the newly formed RNA

Elongation

Three impediments of transcription elongation:

1. 2. 3.

pause arrest termination

Active RNAP II
3OH

Pause RNAP II
3OH

Arrest RNAP II
3OH

TFIIF ELL Elongins CSB DSIF FCP1

TFIIS

10.5 Homeodomain from Engrailed protein interacting with its specific DNA recognition site

DSIF/NELF-mediated checkpoint to ensure pre-mRNA capping

DISF
2 5 2 5 2 5

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

Sequence requirements for 3 end formation

Animals
STOP
Aux enhancer 10-30 nt < 30 nt Poly(A) site Dowstream El

Poly(A)Signal

U rich

AAUAAA conserved

CA

U/GU rich

S. cerevisiae
STOP Eficiency El UAUAUA and related

10-30 nt

Positioning El AAUAAA and related

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

CFI & CFII Cleavage Factors

CFII

AAUAAA
TAA STOP

77
50

64 Rico en GU
Sitio de corte endonucleoltico

CstF Cleavage stimulating Factor

Zhao et al, 1998

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

CFI & CFII Cleavage Factors

CFII

AAUAAA
TAA STOP

Sitio de corte endonucleoltico

Zhao et al, 1998

Formacin y procesamiento del 3 terminal del mRNA en mamferos: corte y poliadenilacin del pre-mRNA

mRNA

AAUAAA
TAA STOP

100 73

30

160

Zhao et al, 1998

Regulacin de la longitud del tracto de poli (A) en mamferos

La regulacin de la longitud del tracto de poli (A) resulta
de la terminacin de la sntesis procesiva del tracto de adeninas, de la estimulacin de su actividad por CPSF PABI y FIP1 y de su inhibicin por las protenas que se unen al CAP y la poli(A) ribonucleasa
100 73 160 30

mRNA FIP1
CPE
CPEBP

(+)
(+)

(+)

Readenilacin citoplsmica

(+)

(-)
5CAP
CBP80 CBP20

Ribonucleasa

POP2

(-)

Polyadenylation machinery 2003

Factor PAP

Processing step Cleavage & poly(A) addition

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

PABII Poly(A) elongation

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

Polyadenylation: A tail of two complexes

Components of cleavage-polyadenylation apparatus know to interact with the RNA pol II transcriptional elongation complex in yeast

Interrelationships between cleavage/polyadenylation factors and other Steps in mRNA Synthesis

10.5 Transcriptional activators are modular proteins composed of distinct functional domains

10.5 Multiprotein complexes form on enhancers

Acetyl transferase activity

Have homologous sequences

Subunits of yeast TFIID and SAGA

Example of a repressor that recruits a histone methylase to repress transcription

Mammals and plants also DNA methylation -> inherited, maintained during replication.

Schematic of p53 pprotein and its interacting proteins

64

110

290

360

393

Transactivation

Proline-rich

Sequence-specific DNA binding domain

Oligomerization

Basic

TBP TAFII 32 TAFII 70 p62(TFIIH) MDM2 RPA Ad E1B 55kDa CBP/p300

Sin3

SV40 LTAg p53 BP1 p53 BP2 HPV 16/18 E6

TBP TFIIH: XPB, XPD CSB RPA BRCA1 c-abl Ad E4orf6

Role of BRCA 1/2 in DNA replication and repair

Eukaryotic RNA Polymerases

ENZYME RNA POL I PRODUCT RNAs RIBOSOMALES: 28S, 5.8S and 18S rRNAs mRNAs snRNAs such as: U1, U2, U4 and U5 snRNAs tRNAs Some snRNAs such as: 7SL RNA, 7SK RNA, U6 snRNA, MRP, H1, Alu and B2 RNAs 5S rRNA

RNA POL II

RNA POL III

RNA POL IVa Gene silencing RNA POL IVb

TAF 110 I TAF 48 TBP TAF 63 I I

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

RNA POL III

TFIID
TAF 170 III TAF 70 TBP III

TFIIIB

Promotores Clase I:
CTCCGAGTCGNNNNNNTGGGCCGCCGG

on

GC

GC

-180

-107

-45

+20

UCE (Upstream Control Element)

CP (Core Promoter)

G-C rich
UCE y CP son 85% idnticos

Promotores Clase III:

Intragnicos
+55 +80
RNA 5S X. laevis

C
tRNAs

Extragnicos
DSE
Distal Sequence Element

PSE

TATA

Proximal Sequence Element

Mixtos

DSE

PSE

TATA

Promotores Clase III:

Estructura de un promotor de tipo II eucarintico

Binding sites for activators that control transcription of a hypothetical gene
Enhancer Promoter-proximal region Core Promoter

-1.96 kb

-1.56 kb

-200 bp

-100 bp TATA

NF1

NF2 p53
NF4 CRE Oct-2

GR NF8 RAR NF10

YY1 C/EBP AP-1

Estructura bsica del ncleo del promotor (100 pb)

on TATA

TATA+ INR-

on
TATA

TATA+ INR+

INR

on TATA- INR+ INR on TATA- INR-

Other Elements of the Core Promoter

TATA

INR

TFIIB recognition element DPE (Downstream Promoter Elements)

TATA BOX DEFINITION

5-TATAAAAG-3

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.

Arabidopsis thaliana TBP2 structure

TBP induces bending on DNA (~120)

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

SIMILITUD A LA HISTONA H2B

A schematic diagram of metazoan TAFII250 (TAF1/CCG1)

Wassarman, D. A. et al. J Cell Sci 2001;114:2895-2902

Ciclo de la Transcripcin
Open complex formation

Pre-initiation complex assembly

Initiation

Termination Promoter clearence

Transcript elongation

Basal Promoter Structure in Eukaryotes

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

Step-by-step Formation of Preinitiation Complex By Class II Factors (1991)

Model for Assembly of Preinitiation Complex

Footprinting to Define Binding Sites (1990)

Footprinting by reaction with hydroxyl radicals (from 1,10phenanthroline-Cu2+) or with DNaseI Protection appears on both strands from TFIIA and TATAbox binding protein (TBP) from TFIID TFIIB gives little further change, but one nucleotide (+10) becomes exposed to DNase I
Experiments used yeast TBP expressed in E. coli

RNA Polymerase Gives Extensive Protection From DNase I

TAFIIs: A Central Component of TFIID

IP to TBP revealed 8 TAFIIs in Drosophila Gel shows recombinant proteins used to assemble TFIID

TAFIIs have two main functions 1. DNA binding 2. Binding other activators They also can have enzymatic activities, like HAT activity

DNA Binding by TAFIIs

DNase footprinting Detecting transcription by primer extension

DNA Binding by TAFIIs

Photocrosslinking using 5-BrdU

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

Structure of RNA Polymerase II

Kornberg and colleagues, 2001 12 subunits in intact RNAP II Rpb4 & 7 are absent from structure Names are (RNA Polymerase B) Rpb1, 2, and 3 are homologous to , , and of bacterial RNAP Rpb1 includes unique, extended Cterminal domain containing 26-52 repeats of YSPTSPS (important later) Rpb5, 6, 8, 10, and 12 are found in all three eukaryotic polymerases

Fig. 10.10

Mediator Complex: an Additional General Transcription Factor and Co-activator

First hint came from squelching, inhibition of one activator by high concentration of another (1988) Isolated as complex of 20 subunits, > 1000 kDa (1994) Cryo-EM structure with RNAP II (2002) Calls into question direct interactions of activators with TAF components of TFIID

TFIIA: Enhances Binding by TBP

Reported to be dispensable in vitro

Essential in yeast
Relieves autoinhibition of TBP Relatively simple: only two subunits

TFIIB: Structure and Function

Just a single polypeptide of 35 kDa Binds after TFIID and TFIIA Essential for RNA polymerase II binding Two domains N-terminal domain binds RNA polymerase C-terminal domain contacts TBP and bent DNA

TFIIBN

TBP TFIIBC Bushnell et al (Kornberg), 2004

TFIIF: Associates Tightly with RNA Polymerase

Cryo-EM (16 ) of RNAPII-TFIIF (2003)

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

Model for Assembly of Preinitiation Complex

TFIIH: Structure and Function

Last general transcription factor to bind in vitro

Plays two major roles Phosphorylates the C-terminal domain of RNA polymerase Unwinds DNA at initiation site to create transcription bubble

TFIIE: Helps TFIIH bind

Phosphorylation of the CTD of RNA Polymerase II

Phosphorylation is stimulated by other factors, notably TFIIE Phosphorylation is even observed of a fusion protein of the CTD with GAL4 transcription factor Two serine residues in repeats are phosphorylated (S2 and S5); S5 is heavily phosphorylated early in elongation and S2 is phosphorylated later

TFIIH: Creation of the Transcription Bubble

Of nine subunits, four are kinase- and five are helicase-associated One helicase subunit, RAD25 in yeast, is essential and has helicase activity

Model for General Transcription Factors: Initiation, Promoter Clearance, and Elongation

Hahn, Nat. Struct. Mol. Biol (2004) 11, 394-403

Retention of a Scaffold Complex Allows Efficient Re-initiation

Subsequent initiation events can be more efficient because most of initiation complex is already formed

Hahn, Nat. Struct. Mol. Biol (2004) 11, 394-403

Models For Termination of Transcription on mRNAs

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.

Class I factors: There Are Only Two

Core binding factor: SL1 in human

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

Common Themes in Eukaryotic Transcription 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

YSPTSPS: 26 yeast 32 C. elegans 45 in Drosophila 52 in mammals

FCP1 phosphatase

2 5 2

SSu72 phosphatase
3 end processing

RNAP II

The phosphorylation cycle of the CTD of the large subunit of 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.

Elongating RNA Polymerase II and size of CTD

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

Newly initiated RNAP II has a tendency to slip laterally

Slip of RNAP II is greatly reduced after RNA-DNA hybrid is 8-9 nt in length

Slip of RNAP II is undetected after RNA is 23 nt long

X-ray studies of co-crystal of RNAP II and TFIIB show that TFIIB impedes the exit path of the newly formed RNA

Elongation

Three impediments of transcription elongation:

1. 2. 3.

pause arrest termination

Active RNAP II
3OH

Pause RNAP II
3OH

Arrest RNAP II
3OH

TFIIF ELL Elongins CSB DSIF FCP1

TFIIS

Human TBP

Ramachandrian plot

Ferenc Mller and Lszl Tora (2004). EMBO J. 23, 2-8

Yeast

Land Plants

C. elegans

Drosophila

Vertebrates

Ferenc Mller and Lszl Tora (2004). EMBO J. 23, 2-8

Ferenc Mller and Lszl Tora (2004). EMBO J. 23, 2-8

Acetyl transferase activity

Have homologous sequences

Subunits of yeast TFIID and SAGA

10.5 Homeodomain from Engrailed protein interacting with its specific DNA recognition site

DSIF/NELF-mediated checkpoint to ensure pre-mRNA capping

DISF
2 5 2 5 2 5

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

Sequence requirements for 3 end formation

Animals
STOP
Aux enhancer 10-30 nt < 30 nt Poly(A) site Dowstream El

Poly(A)Signal

U rich

AAUAAA conserved

CA

U/GU rich

S. cerevisiae
STOP Eficiency El UAUAUA and related

10-30 nt

Positioning El AAUAAA and related

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

CFI & CFII Cleavage Factors

CFII

AAUAAA
TAA STOP

77
50

64 Rico en GU
Sitio de corte endonucleoltico

CstF Cleavage stimulating Factor

Zhao et al, 1998

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

CFI & CFII Cleavage Factors

CFII

AAUAAA
TAA STOP

Sitio de corte endonucleoltico

Zhao et al, 1998

Formacin y procesamiento del 3 terminal del mRNA en mamferos: corte y poliadenilacin del pre-mRNA

mRNA

AAUAAA
TAA STOP

100 73

30

160

Zhao et al, 1998

Regulacin de la longitud del tracto de poli (A) en mamferos

La regulacin de la longitud del tracto de poli (A) resulta
de la terminacin de la sntesis procesiva del tracto de adeninas, de la estimulacin de su actividad por CPSF PABI y FIP1 y de su inhibicin por las protenas que se unen al CAP y la poli(A) ribonucleasa
100 73 160 30

mRNA FIP1
CPE
CPEBP

(+)
(+)

(+)

Readenilacin citoplsmica

(+)

(-)
5CAP
CBP80 CBP20

Ribonucleasa

POP2

(-)

Polyadenylation machinery 2003

Factor PAP

Processing step Cleavage & poly(A) addition

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

PABII Poly(A) elongation

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

Polyadenylation: A tail of two complexes

Components of cleavage-polyadenylation apparatus know to interact with the RNA pol II transcriptional elongation complex in yeast

Interrelationships between cleavage/polyadenylation factors and other Steps in mRNA Synthesis

10.5 Transcriptional activators are modular proteins composed of distinct functional domains

10.5 Multiprotein complexes form on enhancers

9.5 Chromatin exists in extended and condensed forms

Figure 9-29

9.5 The solenoid model of condensed chromatin

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

TATA box now available for binding

ubc

2068

Cinasa
HAT

Cinasa
BD BD

TFIID
Ac Ac

Bromodominios
Acetil lisina DNA

Ub

H1 H1 Nucleosoma ?
P P

N
Ub

H1
Ub

In yeast, histone acetylation can precede or follow remodeling

As a result general TFs can bind, and the gene is transcribed

Acetyl transferase activity

Have homologous sequences

Subunits of yeast TFIID and SAGA

Example of a repressor that recruits a histone methylase to repress transcription

Mammals and plants also DNA methylation -> inherited, maintained during replication.

Schematic of p53 pprotein and its interacting proteins

64

110

290

360

393

Transactivation

Proline-rich

Sequence-specific DNA binding domain

Oligomerization

Basic

TBP TAFII 32 TAFII 70 p62(TFIIH) MDM2 RPA Ad E1B 55kDa CBP/p300

Sin3

SV40 LTAg p53 BP1 p53 BP2 HPV 16/18 E6

TBP TFIIH: XPB, XPD CSB RPA BRCA1 c-abl Ad E4orf6

Role of BRCA 1/2 in DNA replication and repair