7. Transcription
Initiation, elongation, termination
Catalyzed by RNA polymerase
– “Transcription bubble”: DNA transiently separated into
single strands
– One strand is used as a template
– Unwinding point & rewinding point
– Rate ≈ 40 nucleotides/second at 37° for bacteria
RNA polymerase
– Many subunits: catalytic site, CTD with (YSPTSPS)n
– pol I, pol II, pol III
14. Basal transcription apparatus (general
factors & RNA polymerase)
Proximal cis-regulatory module
Distal cis-regulatory modules
Modules = discrete DNA elements that contain specific
sequence motifs with which DNA binding proteins interact
and transmit molecular signals to genes
Promoter
Enhancer
15. BTA
– General factors: TFIIx
– Mechanics of initiating RNA
synthesis at all promoters
– Determines location of
transcription startpoint
– Complex with RNA polymerase
– TATA
• ~ 25bp upstream
• 8bp consensus of A•T pairs
• Tends to be surrounded by
G•C rich regions
• TBP, 11 TAFs : TFIID (~800kD)
18. Promoter
recognition Function = to be recognized by proteins; so differs
from exon, …
Any essential nucleotide sequence should be
conserved
– Some variation is permitted
– When is it sufficiently conserved?
– Idealized sequence with base most often present:
consensus sequence by aligning all known examples
– Only conservation of very short sequences; 60 bp
associated with RNA pol lack conservation
19. Variety of elements can contribute, none is essential
for all promoters (mix & match principle)
CAAT box ~ -80bp GGCCAATCT
– increases promoter strength
– Bound by CTF/NF1 family, CP1 & CP2, C/EBP, ACF
GC box GGGCGG
– SP1
Octamer (8bp) ATTTGCAT
– Bound by Oct1 (ubiquitous): activates histon H2B
– Bound by Oct2 (lymphoid cells): Ig kappa light chain
– context is important
20. Modular nature of the promoter:
– Equivalent regions can be exchanged
– Main purpose = to bring the factors they bind into the
vicinity of the initiation complex
– Protein-protein interactions determine the efficiency of
the initiation reaction
Sequence elements influence the frequency of
initiation
Repression of transcription:
– Generally by influencing chromatin structure
– By repressors, e.g. Dr1/DRAP1 binds to TBP and
CAAT displacement protein (CDP)
23. Modules
50 bp to 1.5 kbp in size
4-8 TFs (often multiple sites); higher density of regulatory
elements than in the promoter
Many elements are common elements in promoters, e.g.
AP1 and the octamer
Can stimulate any promoter placed in its vicinity
Can function anywhere (cfr β-globin: 200 fold in vivo) ;
Position relative to promoter can vary substantially; can
function in either orientation
24.
25. Binding sites for activators that control transcription of the mouse transthyretin (TTR)
promoter in hepatocytes. HNF = hepatocyte nuclear factor. [See R. Costa et al., 1989, Mol. Cell
Biol. 9:1415; K. Xanthopoulus et al., 1989,Proc. Nat’l. Acad. Sci. USA 86:4117.]
Example: TTR
26. Example: muscle specific modules
Creatine kinase, myosin light chain, skeletal actin, myosin heavy chain
27. Example: β-globin
Model for the control of the human β-globin gene. Some of the gene regulatory proteins shown,
such as CP1, are found in many types of cells, while others, such as GATA-1, are present in only
a few types of cells including red blood cells and therefore are thought to contribute to the cell-
type specificity of β -globin gene expression. (Adapted from B. Emerson, In Gene Expression:
General and Cell-Type-Specific [M. Karin, ed.], pp. 116-161. Boston: Birkhauser, 1993.)
30. Current view:
– same sort of interaction with basal apparatus as the
proximal promoter module
– Increase the concentration of transcription factors in the
vicinity of the promoter
Intervening DNA: extruded as a large “loop”
Generality: not yet clear (what proportion of
promoters require an enhancer?)
31. Four activators enriched in
hepatocytes plus the ubiquitous
AP1 factor bind to sites in the
hepatocytespecific enhancer and
promoter-proximal region of the
TTR gene.
The activation domains of the
bound activators interact
extensively with co-activators,
TAF subunits of TFIID,
Srb/Mediator proteins, and
general transcription factors,
resulting in looping of the DNA
and formation of a stable
activated initiation complex.
Cooperative assembly
32. Limited knowledge
Experimentally verified binding sites
Experimentally verified “composite elements” or
CE’s
– GR site + AP-1 in proliferin promoter
– Synergistic: result in non-additively high level
– Antagonistic: overlapping sites, masking an activation
domain,…
– Direct or through coactivator
Few modules characterized that have multiple
elements, some in developmental biology
33. Side-track: Transcription factors
5% of our proteins
Activities controlled in regulatory pathways
Independent domains responsible for activities:
– Recognition of specific target sequences
– Binding to other components
of the transcription apparatus
– E.g. yeast GAL4
34. Protein-DNA interactions
– Proteins with high affinity for a specific sequence also
possess a low affinity for any (random) DNA sequence
– E.g. Lac repressor E. coli: Free:bound = 10-4
– High-affinity site competes with the large number of
low-affinity sites; repressor binds ≈107
times better to
operator DNA (bound 96% of time for 10
molecules/cell)
35. How the different base
pairs in DNA can be
recognized from their
edges without the need to
open the double helix.
36. The binding of a gene regulatory protein to the major
groove of DNA.
Typically, a protein-DNA interface
consists of 10 to 20 such contacts,
involving different amino acids, each
contributing to the binding energy of
the protein-DNA interaction.
38. All of the proteins bind DNA as dimers in which the two copies of the recognition helix (red
cylinder) are separated by exactly one turn of the DNA helix (3.4 nm). The second helix of the
helix-turn-helix motif is colored blue. The lambda repressor and cro proteins control
bacteriophage lambda gene expression, and the tryptophan repressor and the catabolite
activator protein (CAP) control the expression of sets of E. coli genes.
Helix-Turn-Helix
39. Homeodomains
– Related to helix-turn-helix bacterial repressors
– Homeobox = 60 AA residues
– E.g. en, eve, Hox, Oct-1, Oct-2 (Oct also have Pou domain
next to homeodomain)
The homeodomain is folded into three alfa helices, which are packed tightly together by hydrophobic interactions (A). The part
containing helix 2 and 3 closely resembles the helix-turn-helix motif, with the recognition helix (red) making important contacts
with the major groove (B). The Asn of helix 3, for example, contacts an adenine. Nucleotide pairs are also contacted in the minor
groove by a flexible arm attached to helix 1. The homeodomain shown here is from a yeast gene regulatory protein, but it is
nearly identical to two homeodomains from Drosophila, which interact with DNA in a similar fashion. (Adapted from C.
40. Helix-loop-helix (HLH)
– DNA binding (helix) & dimerization
– Class A: ubiquitouslyh expressed proteins, e.g.
E12/E47
– Class B: tissue-specific expression, e.g. MyoD,
myogenin, Myf-5
– Myc proteins (separate class)
Leucine zippers fig 21.15
– Dimerization motif
– E.g. Jun+Fos = AP1
– Gcn4 ->
42. Figure 1 Genome-wide comparison of transcriptional activator families in eukaryotes.
The relative sizes of transcriptional activator families among Homo sapiens,
D. melanogaster, C. elegans and S. cerevisiae are indicated, derived from an analysis of
eukaryotic proteomes using the INTERPRO database, which incorporates Pfam, PRINTS
and Prosite. The transcription factors families shown are the largest of their category out
of the 1,502 human protein families listed by the IPI.
44. Transcription factories
cfr. replication factories
Active RNA polymerases are concentrated in discrete
'factories' where they work together on many different
templates
Complexes for transcription and RNA processing are
likely to be immobile structures within the gel-like
nucleoplasm (Burns et al, 2001; Kimura et al, 1999)
Transcriptional interference: phenomenon where
transcription of one gene prevents transcription of an
adjacent gene. Discovery: Cells were transfected with a
retroviral vector encoding resistance to neomycin and
azaguanine, and clones harboring a single copy of the
vector selected. Expression of the 3' gene was
suppressed when selection required expression of the 5'
gene, and vice versa. In addition, hardly any cells grew
in both neomycin and azaguanine
45. Cook, 1999 (Science)• Enhancers
•dynamic equilibrium
•enhancing the probability of the key transcription cycle interactions
•Element 5’ or 3’ doesn’t matter!
46. Recap: evolution of understanding of
eukaryotic transcription
Lemon and Tjian, Genes Dev. 14: 2551-2569 (2000)
49. Activate/inactivate a TF
Transport through nuclear pores from cytoplasm
to nucleus (e.g. masking NLS, nuclear localization
signal, can regulate this transport)
Link to Ubiquitin protease system
– Rapid turnover of promoter bound TF: resets signaling
pathway: cell can continuously monitor its environment
Tissue-specific synthesis
– Development, e.g. homeodomain proteins
Modification
– Phosphorylation, acetylation, methylation
– E.g., AP1 (= Jun+Fos) → active form by
phosphorylation
– E.g., p53 acetylated (modulates interactions with
coactivator and repressor proteins
50. Ligand binding
– E.g. Steroid receptors
– Influence: localization or DNA-binding ability
Cleavage
Inhibitor release
– E.g. NF-κB + I- κB (release in B lymphocytes)
Change of partner (active partner displaces
inactive partner)
53. Level 1 = active/inactive factor
Level 2 = cooperation of multiple factors within a
module (all present and active, and all repressors
inactive or absent)
Level 3 = multiple autonomous modules per gene
– Each module can independently activate the gene
– Each has a specific function (e.g. activation in certain
cell type or at particular stage in dvl)
– different circuits of regulation, e.g. metallothionein
gene (MT): heavy metals and steroids, fig 21.1
– Gene can respond to multiple signaling pathways
– Facilitates fine-tuning of transcript levels
54. Combinatorial and context dependent
regulation of transcription
– one factor can induce transcription of one gene
while repressing that of another
55. Example: eve
Experiment demonstrating the modular
construction of the eve gene regulatory region.
(A) A 480-nucleotide-pair piece of the eve
regulatory region was removed and inserted
upstream of a test promoter that directs the
synthesis of the enzyme β-galactosidase (the
product of the E. coli lacZ gene). (B) When this
artificial construct was reintroduced into the
genome of Drosophila embryos, the embryos
expressed β-galactosidase (detectable by histo-
chemical staining) precisely in the position of the
second of the seven eve stripes (C).
(Metamerization)
-
+
57. Principles for specification
1. cis-regulatory transformation of input patterns into
spatial domains of differential gene expression
2. Always assemblages of diverse target sites because
multiple inputs are required
3. Output=novel with respect to any one of the incident
inputs + more precise in space and time => “information
processing”
4. Every specific type of interaction that can be detected in
vitro is fundamentally significant (it is unlikely that highly
specific site clusters, which are of improbable random
occurrence would have no function)
5. Negative & positive inputs
(Davidson, 2001)
58. Cis-regulatory logic device
endo16 of Strongylocentrotus (zee-egel)
Secreted embryonic gut protein
“hardwired biological computational
device”
59.
60.
61.
62. Overview
Gene expression
Initiation of transcription
Regulation of transcription
Influence of chromatin structure
Oncogenes
Techniques
63. Chromatin
Eukaryotic genomes are
packaged with chromatin
proteins
Heterochromatin (highly
condensed, untranscribed)
Euchromatin (more
accessible, transcribed)
Each cell: unique pattern of
heterochromatin and
euchromatin
65. Chicken and egg scenario
TF binding requires chromatin decompaction by
certain factors but the latter also need to interact
with DNA
Solution: probably some TFs can bind to their
recognition sequences even when they are
packaged (e.g. glucocorticoid receptor: only
contacts DNA on one side ⇔ NF1 surrounds
double helix)
67. 2. Histone-modifying complexes
• Phosphorylation, methylation, acetylation
• Histone acetyltransferase (HAT), histone
deacetylase (HDAC)
• How do they impact the structure of the template
and the ability of the transcription machinery to
function?
• lowered positive charge on acetylated N termini,
lowered stability of interaction with DNA
• Disrupting internucleosomal interactions
• Recruiting additional TFs
• A lot of combinatorial possibilities: histon code?
69. Model of the protein
interactions and functions
of the Myc/Max/Mad
transcription network.
Myc-Max and Mad-Max (along with Mnt-Max and Mga-Max) complexes bind to DNA to E-boxes. Binding can be
affected by the context, sequence, cooperativity, and location of the E-boxes. Myc-Max heterodimers activate
transcription by recruiting HAT's via TRRAP. This leads to the acetylation of histone tails and the opening of local
chromatin structure. Additionally, Myc-Max appears to repress transcription through Inr elements via an undefined
mechanism. As a result of these activities at target genes, Myc affects proliferation, cell cycle, growth, immortalization,
and apoptosis. When deregulated, Myc cooperates with other oncogenes to cause a variety of cancers.
Mad-Max and Mnt-Max heterodimers repress transcription by recruiting HDAC's via mSin3A. This leads to the
deacetylation of histone tails and the closing of local chromatin structure. As a result of target gene repression, Mad
causes an increased cell doubling time, growth arrest, and the maintenance of differentiation.
Grandori C, Cowley SM, James LP,
Eisenman RN.
Annu Rev Cell Dev Biol. 2000;16:653-
99
70. Cytosine methylation
mCG often in inactive vertebrate genes
After replication of methylated DNA,
methyl groups are added to daughter strands
CpG islands
imprinting
71. Imprinting
Imprinted genes are genes whose expression is determined by the
parent that contributed them.
Imprinted genes violate the usual rule of inheritance that both alleles in
a heterozygote are equally expressed.
Examples of the usual rule:
– If a child inherits the gene for blood group A from either parent and the
gene for group B from the other parent, the child's blood group will be
AB.
– If a child inherits the gene encoding hemoglobin A from either parent and
the gene encoding hemoglobin S from the other parent, the child's red
blood cells will contain roughly equal amounts of the two types of
hemoglobin.
But there are a few exceptions to this rule. A small number of genes in
mammals (~50 of them at the most recent count) have been found to
be imprinted. Because most imprinted genes are repressed, either
– the maternal (inherited from the mother) allele is expressed exclusively
because the paternal (inherited from the father) allele is imprinted or
– vice-versa.
74. Consistent correlation between gene
silencing (e.g. in B en T lymphocytes) and
presence in heterochromatin regions
– LCR, enhancers, insulators: act by maintaining
endogenous loci in a chromatin compartment
that is either transcr. permissive or
nonpermissive?
75. Position variegation
Position effects can be observed for the Drosophila white gene. Wild-type flies
with a normal white gene have red eyes. If the white gene is inactivated by
mutation, the eyes become white (hence the name of the gene). In flies with a
chromosomal inversion that moves the white gene near a heterochromatic
region, the eyes are mottled, with red and white patches. The white patches
represent cells where the white gene is silenced and red patches represent cells
that express the white gene. (After L.L. Sandell and V.A. Zakian, Trends Cell Biol.
2:10-14, 1992.)
76. Overview
Gene expression
Initiation of transcription
Regulation of transcription
Alteration of chromatin structure during
transcription
Oncogenes
Techniques
77. The development and metastasis of human
colorectal cancer and its genetic basis.
A mutation in the APC tumor-suppressor gene in
a single epithelial cell causes the cell to divide,
although surrounding cells do not, forming a
mass of localized benign tumor cells called a
polyp. Subsequent mutations leading to
expression of a constitutively active Ras protein
and loss of two tumor-suppressor genes, DCC
and p53, generates a malignant cell carrying all
four mutations; this cell continues to divide and
the progeny invade the basal lamina that
surrounds the tissue. Some tumor cells spread
into blood vessels that will distribute them to
other sites in the body. Additional mutations
cause exit of the tumor cells from the blood
vessels and growth at distant sites; a patient with
such a tumor is said to have cancer. [Adapted
from B. Vogelstein and K. Kinzler, 1993, Trends
Genet. 9:101.]
78. Overview
Gene expression
Initiation of transcription
Regulation of transcription
Alteration of chromatin structure during
transcription
Oncogenes
Techniques
82. Sources
B Lewin, Genes VII
Lodish et al. Molecular Cell Biology
EH Davidson: Genomic Regulatory
Systems
Alberts et al. Essential Cell Biology
EM Blackwood & JT Kadonaga: Going the
distance: a current view of enhancer action
Cell, February 22, 2002: 108 (4) "Reviews
on Gene Expression"
Notes de l'éditeur
RNA pol I: rRNA RNA pol II: mRNA RNA pol III: tRNA e.a. small RNAs
Transcription-control regions Signals for 3’ cleavage and polyadenylation Signals for splicing of primary RNA transcripts Mutations in these signals prevent expression of a functional mRNA and thus of the encoded protein
Different Sigma factors
Activator proteins communicate with the basal transcription machinery at promoters through intermediary factors. Two candidates for such factors have been identified, the TAF complex which interacts with TBP, and the Mediator complex which interacts with the RNA polymerase II CTD. TAFs enable a response to activators in a partially reconstituted Drosophila or human transcription system, while Mediator supports activation in a fully defined yeast system. Deletion or destruction of TAFs has, however, no effect upon induction or transcription of most genes in yeast in vivo. Inactivation of Mediator components, on the other hand, abolishes both induction of specific genes and transcription in general. It therefore appears that Mediator is the primary conduit of information from enhancers to promoters in vivo. A small number of yeast promoters do require TAFs for transcription in vivo, including those for G1 cyclins and for some cell cycle-independent genes. Dissection of these promoters identifies sequences surrounding the TATA box and not upstream activating elements as responsible for the TAF requirement. TAFs are evidently involved in promoter selection and specificity rather than enhancer-promoter interaction.
GAL80 prevents GAL4 from activating transcription: GAL80 is released when galactose is present, thus allowing GAL4 to activate its target genes
Repressors cancel the output of the activators
Drosophila, rho = Cell surface component, Signaling Neuroectodermal territory->ventral CNS Repression by Snail in the mesoderm mutated snail sites -> no more repression new snail sites somewhere else -> OK autonomous: 2 modules linked (rho element + eve stripe 2: Kr repressor, Bcd activator) -> A/P rho pattern + D/V eve stripe 2 2 x represson element of zen (with Dl sites) gene placed distal to the eve stripe 2 Deadringer & Cut corepressors interact at I
A1. Initial: vegetal plate of blastula-stage embryo -> endo+mesodermal cell types (but no skeletogenic)
