Download Transcription Factors (from Wray et al Mol Biol Evol 20:1377)

Transcription
The Gene
• Complex collection of sequences that
o Controls a phenotype
 Individually
 OR
 Complexed with action of other genes
• Size varies
• Structural features vary
• Encode for a protein(s) that is translated from a mRNA
• Expression
o Requires many associated factors
Transcription - the synthesis of RNA from a DNA template
Transcription, whether prokaryotic or eukaryotic, has three main
events.
1. Initiation
• Binding of RNA polymerase to double-stranded DNA
• This step involves a transition to single-strandedness in the region
of binding
• RNA polymerase binds at a sequence of DNA called the promoter
Initiation is the most important step in gene expression!!!
2. Elongation
• The covalent addition of nucleotides to the 3' end of the growing
polynucleotide chain
• Involves the development of a short stretch of DNA that is
transiently single-stranded
3 Termination
• The recognition of the transcription termination sequence and
• Release of RNA polymerase
Eukaryotic RNA Polymerase
1. Three types exist
Type of Polymerase
RNA Polymerase I
RNA Polymerase II
RNA Polymerase III
Product
rRNA
hnRNA
tRNA
Location
nucleolus
nucleoplasm
nucleoplasm
2.Protein is greater than 500 kd in size
3. Multiple components
• Two large subunits
• <10 small subunits
4. Many non-polymerase factors required for binding of the enzyme to
DNA
Product of Transcription
Transcription Unit
• Extends from the promoter to the termination sequences
Upstream Sequences
• Sequences before the start site
Downstream Sequences
• Sequences after the start site
Primary Transcript.
• Immediate transcription product
Steps in Model Eukaryotic Transcription
Adenovirus late promoter
• Requires four accessory factors and RNA Polymerase II added in a
defined manner
Order Factor
Length of promoter covered (bp)
1.
TFIID
-42 to -17 (binds TATA box)
2.
TFIIA
-80 to -17
3.
TFIIB
-80 to -17 and -10 to +10
4.
RNA Polymerase II
-80 to +15
5.
TFIIE
-80 to +30
The Transcription Product
Heterogeneous nuclear RNA
• hnRNA
• Complexity of hnRNA is 4x the mRNA pool
o Why??
 Splicing of introns from the primary transcript
o Average size
 8000 - 10,000 nucleotides
o Range
 2000 - 14,000 nucleotides
Splicing
• Removes the introns from the hnRNA
• Alternate splicing
o An intron is skipped; or
o Uses signals other than the GT/AG associated with introns
signals
o Result
 Multiple transcripts and proteins from a single gene
sequence
Finishing the mRNA
A. 5' Capping
• Protects the transcript
o Occurs so quickly that we rarely see the original 5' base of
the message
• Unique nucleotide
o 5' methyl guanosine
• Added immediately after the start of transcription
• Sequence linkage
o 5' methyl guanosine 5'-5' linkage
 Not the typical 5'-3' linkage
• Enzyme
o Guanylyl transferase.
B. 3' Polyadenylation
• Adds a Poly-A tail (many adenines) to end of transcript
• Found in all eukaryotic mRNA
• Sequence signal for adding the poly-A tail
o 5'-AAUAAA-3'
• Located
o About 10-30 bp upstream of the poly A tail.
Transcription Factors General Terms and Concepts
Promoter
• Difficult to define
• General definition
o All the DNA sequences containing binding sites for RNA
polymerase and the transcription factors necessary for normal
transciption
Transcription Factor
• Any protein other than RNA polymerase that is required for
transcription
Functions of Transcription Factors
• Bind to RNA Polymerase
• Bind another transcription factor
• Bind to cis-acting DNA sequences
Basal Transcription Apparatus
• RNA polymerase + General transcription factors
• Both needed to initiate transcription
Upstream Transcription Factors
• Ubiquitous factors that increase the efficiency of transcription
initiation
• Set of factors unique to each promoter
Inducible Transcription Factors
• Act in the same manner as an upstream factor
o BUT
 Their synthesis is regulated in a temporal or spatial
manner
Transcription Factors
(from Wray et al Mol Biol Evol 20:1377)
Phenotype is affected by mutations in:
1. Structural region of a gene
• Function of a protein is modified (structure/function relationship)
2. Regulatory region of a gene
• When the protein is expressed (gene regulation)
Considerations of gene regulations
1. Change the regulation pattern of a gene can change phenotype
2. One transcription factor (TF) can affected multiple genes in a pathway
3. TF orhtologs will regulate different organisms differently
4. Promoter contain module that can be changed to affect expression
Approaches to studying gene regulation
1. Mutants
Do induced mutants represent natural variation?
2. Expression patterns
• Expression patterns of orthologs can differ among species
3. Expression levels
• Phenotypic differences can result from changes in the amount of a
protein
Effect of varying expression level
1. Spatial effects
• Varying the amount of expression in a tissue can change phenotype
2. Cis-effects
• Variation in expression level often related to changes in ciselements
3. Inducibility
• Alleles can be induced differentially
Levels of expression can vary at the:
1. mRNA level
2. Protein level
What amount of the gene expression variation is the
result of “controlling region” variation???
1. Natural variation exists in promoters
• Associated with phenotypic changes
2. Artificial selection of promoter sequences can change expression
• Maize tb locus is an example
3. Promoter “elements” are conserved among species
• Specific sequences important for gene expression
4. Variation in promoter sequence related to human disease
susceptibility
• Susceptibility to specific pathotypes related to promoter sequences
Transcription patterns are variable
1. Transcription initiation is the most important step in phenotypic
expression
2. Regulation is at the gene not gene family level
• Paralogs are independently regulated
3. Transcription is dynamic
• Expression levels vary
• Expression can fluctuate rapidly
• Expression in neighboring cells can differ
4. Expression profiles vary among genes
• Regulatory gene expression profile is inducible and highly variable
• Housekeeping gene expression is generally constitutive but varies
in response to stimuli and by cell type
Role of Controlling Regions (=Promoters) in Gene
Expression
1. Promoters
• Contain sequence motifs that bind factors that modulate gene
expression
2. Constitutive (housekeeping) promoters
• On by default,
• Turned off in response to stimuli
3. Inducible promoters
• Off by default
• Turned on in response to stimuli
4. TF determine if genes are turned on or off
Promoters
1. Universal conserved features are not found
2. Common sequence motifs not found
Basal Gene Expression
1. Basal promoter
• RNA polymerase complex binding site
• Contains TATA box or initiator element
• Null promoter exist
• Lacks TATA box or initiator element
• Multiple basal promoters exist for some genes
2. TATA-box binding protein (TBP)
• First protein to bind the basal promoter
• Other proteins guide TBP to the binding site
3. RNA polymerase holoenzyme complex
• Complex interactions of proteins builds the transcription complex
4. Transcription start site
• Begins about 30 bp downstream of site where the transcription
complex
5. Translation start site
• Begins about 10 – 10,000 bp from transcription start site
6. Basal promoters provides for minimal, low level of expression
• Expression mediated by constitutively expressed general
transcription factors
Modifying Basal Gene Expression Levels
1. TF binding to controlling regions required for full gene expression
• TF are specific to cell types and stimuli conditions
• Interaction of controlling regions and TF controls gene expression
Controlling Region TF Binding Sites
1. Binding sites are isolated in controlling region
• Binding sites are embedded in regions to which no TF bind
2. Binding sites numbers
• 10 – 50 binding sites for 5 –15 TF
3. Role of other sequences
• Local, sequence-specific conformational changes can affect TF
binding
• AT-rich regions
• Z-DNA
4. Spacing of binding sites
• Partial overlap
• 10s of kilobases
Features of TF Binding Sites
1. Size
• Footprint (sequences covered by TF) is 10-20 bp
• Direct binding site is 5-8 bp
• Essential sequence is 4-6 bp
2. Site definition
• Consensus sequence (although not all consensus sequences bind
TF)
• Biochemical activity (required to define a functional sequence)
3. Binding sites can overlap
• TF pool determines which site is bound
• Binding sites compete for a limited TF pool
4. Location
• 100 basepairs to 100 kilobases from transcription start site
5. Functional location000
• >30 kb 5’ of basal promoter
• few kb of basal promoter
• in 5’ UTR
• introns
• >30 kb 3’ of basal promoter
• exon
• other side of adjacent gene
Features of TF Binding Sites (cont.)
6. Location constraints
• Some sites are constrained to specific positions relative to
transcription start site
7. Isolating binding sites effects
• Insulator sequences limit TF interactions to specific basal
promoters
• TATA or TATA-less TF interaction specificity
• Specific recruitment of TF at a specific sequence to interact with
basal promoter
8. Multiple control
• On set of binding sets controls paralogs on opposite strands in
opposite orientation
• Cross regulated sites share common binding sites
Abundance of Transcription Factors
1. TF are members of small to large multi-gene families
• Arabidopsis
• (CCAAT-DR1 Family) to 164 (C3H Family) paralog families
3. Result from gene duplication events
4. 12-15 unique DNA binding domains
• Evolutionary conservation
Modular Nature of Transcription Factors
1. DNA binding domain
• Localized
o MADS-box or homeo domains
• Dispersed
o Zn-finger or leucine zipper domains
2. Protein-protein interaction domain
• Binding to other proteins necessary for activation
3. Intracellular trafficking domains
• Nuclear localization signal
4. Ligand binding domain
• Steroid or hormone-binding domains
5. Evolutionary domain shuffling has occurred
• Protein-protein interaction domain lost but DNA binding domain
maintained
Transcription Factor DNA Binding Domain
1. Most bind the major groove of DNA
2. Domain sequence is highly conserved
• Single amino acid mutations can alter significantly TF binding
3. TF binding specificity ranges from 3-5bp
4. Specificity may be increased by
• Multiple binding domains
• Domains that bind minor groove
• Dimerization of two proteins, either homomeric or heteromeric
5. Binding is strong and highly specific
• 5000 – 20,000 copies of TF needed for high binding specificity
6. Cofactor interactions increase specificity
• Phosophorylation
7. Paralogs may have unique binding specificities
Transcription Factor Protein-Protein Interactions
Modulate Gene Expression
1. Increase (or decrease) the frequency in which the transcription
apparatus is built
• Can recruit (or prevent recruitment) of apparatus components
2. Specific interactions necessary for effects to be realized
• As homodimers
• As heterodimers
• As solo proteins
3. Neighboring effects
• TF at one site can prevent cofactor from interacting with a
neighboring site
4. Altering chromatin structure
• Recruit other complexes that
• Acetylate, deacetylate, methylate, or demethylate histones
• Methylate or demethylate DNA
5. Create physical bends
• Facilitates binding of other TF
6. Cofactors can bring TF and transcriptional apparatus together
Transcription Factor Activation or Repression of
Transcription
1. Activation or repressor domains exists in TF
2. Action can be mediated through direct (or indirect via TAF)
interaction with TBP
Transcription Factor Activation Depends Upon
Specific Modifications and Interactions
1. Post-translational modifications such as phosphorylation necessary
2. Activation and repression domains may reside in same protein
• Specific functional activity depends upon cofactor involvement
3. A TF can act as a repressor if it blocks the binding site of a TF
activator
• This interaction can have a downstream effect on other expression
steps
Cooperative-binding and Interaction of Transcription
Factors
1. Precise spacing required for some interactions involving TF
• Nucleosome (40 bp multiples) or decondensed DNA (10 bp
multiples) distances
• Interactions with chromatin remodeling complexes may have a
moderate distance requirement
2. Bending and looping supports interactions
• Removes distance specificity requirement
Role of Functional Modules
1. Functional modules can have several functions
• Initiate transcription
• Boost transcription rate
• Mediate extracellular signals
• Repress transcription
• Insulate on module from another (insulator function)
• Bring other modules into contact with basal promoter
• Integrate other module status into a global expression pattern
Additive and Epistatic Interactions of Transcription
Factors
1. Modifying one TF and its module interaction can additively reduce
the phenotype
2. Modifying insulator, tethering, or inegrator TF functions is epistatic
3. Proper expression, recruitment, and modular association of TF is
necessary for full phenotypic expression
A Transcription Family Has Multiple Target Genes
1. Because of the limited number of TF, a single TF may interact with
10s to 100s of genes
2. Drosophila eve and ftz regulate the majority of genes in the genome
3. The function of TF networks may genes
4. Mutations can be modulated by the effects of other downstream genes
The Genome Is Significantly Involved in Gene
Regulation
1. The number of promoter sequences is equal to the number of protein
coding sequences
2. Transcription regulation a major function of the genome
Distribution of Transcription Factors Among Dicot Genomes
(family assignment rules from: http://planttfdb.cbi.pku.edu.cn/)
Family
AP2
ARF
ARR-B
B3
BBR-BPC
BES1
C2H2
C3H
CAMTA
CO-like
CPP
DBB
Dof
E2F/DP
EIL
ERF
FAR1
G2-like
GATA
GRAS
GRF
GeBP
HB-PHD
HB-other
HD-ZIP
HRT-like
HSF
LBD
LFY
LSD
M-type
MIKC
MYB
MYB_related
NAC
NF-X1
NF-YA
NF-YB
NF-YC
NZZ/SPL
Nin-like
RAV
S1Fa-like
SAP
SBP
SRS
STAT
TALE
TCP
Trihelix
VOZ
WOX
WRKY
Whirly
YABBY
ZF-HD
bHLH
bZIP
Total
Grape
(3x)
19
17
12
29
5
6
64
43
4
6
6
7
22
7
2
80
18
40
19
43
8
1
2
7
33
1
19
44
1
3
18
36
138
57
71
3
7
17
8
1
8
1
2
1
19
5
1
21
15
26
2
11
59
2
7
10
115
47
1276
Papaya
(3x)
17
10
12
34
3
6
76
28
4
9
4
6
20
6
4
77
19
51
23
42
7
4
1
8
29
2
18
35
1
2
225
20
98
51
82
1
5
11
4
1
6
2
1
2
11
4
1
11
22
29
2
11
49
2
9
10
105
46
1379
Arabidopsis
(3x + 2x)
30
37
21
77
17
14
116
66
10
22
9
14
47
16
6
139
26
64
41
37
9
23
3
11
58
2
25
50
1
12
70
76
168
97
138
2
21
27
21
1
17
7
4
1
30
16
4
33
33
34
3
18
90
4
8
18
225
127
2296
Tomato
(3x + 3x)
27
22
21
73
6
9
99
48
7
13
4
10
33
8
9
137
28
59
30
54
13
11
2
16
58
1
26
47
1
3
67
32
140
79
101
2
10
29
20
1
10
3
1
3
17
9
1
21
36
31
2
10
81
2
9
22
161
70
1845
Soybean
(3x + 2x + 2x)
76
85
42
112
22
19
267
136
23
32
19
36
93
28
12
330
103
164
70
139
31
11
11
31
140
1
61
111
2
17
88
160
369
265
247
8
57
46
35
0
45
5
4
2
73
33
1
101
71
93
20
42
233
13
34
54
480
266
5069
Distribution of Transcription Factors Among Monocot Genomes
(family assignment rules from: http://planttfdb.cbi.pku.edu.cn/)
Family
AP2
ARF
ARR-B
B3
BBR-BPC
BES1
C2H2
C3H
CAMTA
CO-like
CPP
DBB
Dof
E2F/DP
EIL
ERF
FAR1
G2-like
GATA
GRAS
GRF
GeBP
HB-PHD
HB-other
HD-ZIP
HRT-like
HSF
LBD
LFY
LSD
M-type
MIKC
MYB
MYB_related
NAC
NF-X1
NF-YA
NF-YB
NF-YC
NZZ/SPL
Nin-like
RAV
S1Fa-like
SAP
SBP
SRS
STAT
TALE
TCP
Trihelix
VOZ
WOX
WRKY
Whirly
YABBY
ZF-HD
bHLH
bZIP
Total
Japonica
rice (2x
22
48
11
65
7
6
135
74
7
21
20
13
37
10
11
163
133
62
32
69
19
13
1
17
61
1
38
39
2
12
35
61
130
106
170
2
25
16
19
0
15
4
2
0
29
6
1
45
23
40
2
17
128
2
15
15
211
140
2408
Brachypodium
(2x)
29
36
9
45
4
7
93
53
10
14
11
11
27
7
6
120
69
61
30
48
14
15
5
12
43
1
26
24
1
7
24
51
98
77
109
1
12
17
15
0
15
4
2
0
18
5
1
30
21
32
2
9
87
2
13
15
158
95
1751
Sorghum
(2x)
32
33
13
86
6
9
122
55
10
14
12
11
35
13
10
165
62
56
34
86
11
15
3
8
47
1
25
36
1
6
46
47
132
116
141
3
16
16
18
0
16
4
2
0
22
6
1
28
21
36
2
12
110
2
10
18
233
123
2198
Corn
(2x + 2x)
54
62
13
77
9
16
179
111
10
18
17
20
51
24
9
205
25
89
54
104
32
29
4
28
97
0
49
60
4
20
47
90
203
169
190
4
36
28
25
0
23
3
5
0
55
11
2
52
52
59
10
30
163
6
31
26
308
218
3316
Arabidopsis
(3x + 2x)
30
37
21
77
17
14
116
66
10
22
9
14
47
16
6
139
26
64
41
37
9
23
3
11
58
2
25
50
1
12
70
76
168
97
138
2
21
27
21
1
17
7
4
1
30
16
4
33
33
34
3
18
90
4
8
18
225
127
2296
Defining Transcription Factors – Based on Conserved Pfam
Sequence Motifs (mostly)
(Pfam: accepted motif sequence definitions;
http://pfam.sanger.ac.uk/)
Distribution of Transcription Factor Families between Common
Bean (Phaseolus vulgaris) and Soybean (Glycine max). Soybean has
undergone a genome duplication since its split from common bean.
(family assignment rules from: http://plntfdb.bio.uni-potsdam.de/)
TF family
ABI3VP1
Alfin-like
AP2-EREBP
ARF
ARID
ARR-B
AUX/IAA
BBR/BPC
BES1
bHLH
BSD
bZIP
C2C2-CO-like
C2C2-Dof
C2C2-GATA
C2C2-YABBY
C2H2
C3H
CAMTA
CCAAT
Coactivator p15
CPP
CSD
DBP
DDT
E2F-DP
EIL
FAR1
FHA
G2-like
GeBP
GNAT
GRAS
GRF
HB
HMG
HRT
HSF
IWS1
Jumonji
LFY
LIM
Pv count
41
24
179
27
12
15
30
5
7
155
10
78
8
42
32
8
10
44
8
55
3
6
5
2
11
7
7
25
19
49
5
38
55
10
119
9
1
30
10
21
1
9
Gm count
90
38
363
60
26
31
66
18
16
359
24
204
26
81
64
18
62
153
15
253
9
20
8
4
20
16
12
80
39
131
19
58
119
24
203
24
1
52
22
40
8
20
Ratio
2.2
1.6
2.0
2.2
2.2
2.1
2.2
3.6
2.3
2.3
2.4
2.6
3.3
1.9
2.0
2.3
6.2
3.5
1.9
4.6
3.0
3.3
1.6
2.0
1.8
2.3
1.7
3.2
2.1
2.7
3.8
1.5
2.2
2.4
1.7
2.7
1.0
1.7
2.2
1.9
8.0
2.2
TF family
LOB
LUG
MADS
MBF1
MED6
MED7
mTERF
MYB
MYB-related
NAC
NOZZLE
OFP
PBF-2-like
PHD
PLATZ
Pseudo ARR-B
RB
Rcd1-like
RWP-RK
S1Fa-like
SAP
SBP
SET
Sigma70-like
SNF2
SOH1
SRS
SWI/SNF-BAF60b
SWI/SNF-SWI3
TAZ
TCP
Tify
TIG
TRAF
Trihelix
TUB
ULT
VARL
VOZ
WRKY
zf-HD
Zn-clus
Total
Pv count
49
5
78
3
1
1
34
141
68
90
5
20
3
32
14
6
1
2
12
3
1
23
44
9
37
1
10
18
5
4
27
13
5
22
41
10
1
3
5
90
19
0
2188
Gm count
95
12
180
4
1
3
58
291
314
186
6
47
7
270
34
12
3
8
28
12
2
47
82
13
64
2
22
31
9
5
56
33
1
56
73
24
11
6
8
186
57
0
5225
Ratio
1.9
2.4
2.3
1.3
1.0
3.0
1.7
2.1
4.6
2.1
1.2
2.4
2.3
8.4
2.4
2.0
3.0
4.0
2.3
4.0
2.0
2.0
1.9
1.4
1.7
2.0
2.2
1.7
1.8
1.3
2.1
2.5
0.2
2.5
1.8
2.4
11.0
2.0
1.6
2.1
3.0
Structural variation in Arabidopsis thaliana basic helix-loop-helix
transcription factors (The Plant Cell (2003) 15:1749)
Figure 3. Intron Distribution within the bHLH Domains of the AtbHLH Proteins. Scheme of the intron
distribution patterns (color coded and designated A to I) within the bHLH domains of the AtbHLH proteins. Introns
are indicated by triangles and numbered (1 to 3) based on those present in the bHLH region of PIF3, which is shown
at top. When the position of the intron coincides with that found in PIF3, the intron number is given above the
triangle. For patterns F, G, and H, no intron number above the triangle indicates that the location of the intron within
the bHLH domain is different from that found in PIF3. The percentage of proteins with each pattern is given at right.
The correlation of intron distribution patterns and phylogenetic subfamilies is provided in Figure 2 (central column,
color coded), and the chromosomal distribution of intron patterns is provided in Figure 4 (colored ovals adjacent to
each entry number).
Distribution of the basic helix-loop-helix transcription factory
family in Arabidopsis thaliana (The Plant Cell (2003) 15:1749)
Figure 4. Chromosomal Locations, Intron Distribution Patterns, and Duplication Events for AtbHLH Genes.
Deduced chromosomal positions of the AtbHLH genes are indicated by EN (assigned in Figure 1). Segmentally
duplicated regions in the chromosomes (Chr I to V) are indicated by boxes of the same color (adapted from TIGR).
The total number of bHLH genes per chromosome is indicated at the top of each chromosome in parentheses. The
scale is in megabases (Mb) and is adapted from the scale available on the TIGR database (see Methods). The small
colored ovals at left of the ENs indicate the intron distribution patterns within each gene. The color code
corresponds to the intron patterns shown in Figure 3. Connecting lines (blue and pink) mark the specific cases in
which there is a strong correlation between duplicated genomic regions and the presence of bHLH genes with both
closely related predicted amino acid sequence (close ENs) and the same intron pattern. The blue lines link cases
associated with apparent intrachromosomal duplications (see supplemental Figure 7B online), and the pink lines link
cases associated with apparent interchromosomal duplications (for more details, see supplemental Figure 7C online).
Multiple members of a gene family (rbcS: small subunit of
RUBISCO) have different cis-acting elements (The Plant Cell (1991)
3:1305)
Conserved cis-acting element
Family member specific cis-acting elements
Transcription factors bind to different domains of a promoter in
different tissues. (The Plant Cell (1991) 3:1305)