Chapter 7
some promoters require chromatin-remodeling complexes which
(eg. SWI/SNF in yeast) facilitate "sliding" of nucelosomes and accelerate binding of transcription factors -also required for many other processes involving DNA replication recombination and repair
Transcription Factor-DNA interaction detected by Footprinting
-incubate protein sample with a radiolabeled DNA fragment containing a putative control element (only 5'end is tagged) -add small amounts of endonuclease DNase I to produce about one random cut/DNA fragment -if present in sample, the corresponding transcription factor binds to its site and protects it from endonuclease cuts -DNA fragments are then separated by Gel electrophoresis (fragment size indicates distance from labeled ends). -tells you whether transcription factors are there, when proteins bound cut will not occur ig binds those fragments that originated from random cutting will not appear anymore because they are protected from the protein
promoter-localized chromatin decondensation
1. GCN4 transcription factor binds to USA upstream control element 2. activation domain of GCN4 binds recruits a complex with acetylase activity (facilitates polII assembly at the TATA box)
Three main classes of promoter elements that are recognized by pol II
1. TATA boxes 2. Initiator Sequences 3. CpG islands
promoter-localized chromatin condensation
1. UME6 transcription factor binds to USR1 upstream control element 2. repressor domain of UME6 recruits protein complex that has deacetylase activity (promotes localized nucleosome condensation and prevents pol II from binding to TATA box).
model of telomere sequence silencing in yeast
1. telomere repeat sequences bind to multiple copies of the Rap1 protein 2. Rap1 recruits a complex of Sir proteins one of which displays deactylase activity (SIR 2) 3. Removal of histone acetyl groups (negative charges) from neighboring nucleosomes promotes chromatin condensation.
C2H2 zinc finger
23-22 aa long consensus sequence ex. GL-1 in plants
C4 zinc finger
55 or 56 aa long consensus sequence ex. glucorticoid receptor
the last step of purification of transcription factors uses
DNA affinity column chromatography -DNA control element is covalently linked to the column -the corresponding transcription factor binds to the column under physiological salt concentration while contaminating protein flow through -the transcription factor is recovered by raising the salt concentration to disrupt binding
amino acid identity highest in
DNA binding domain (42-94%) then ligand binding domain (15-57%) last variable region 0%
main control element for transcription
DNA binding protein
maintenance of epigenetic changes requires specific
DNA methyltransferase enzymes and protein complexes that bring histone modifications activities to specific sites sometimes with the help of non-coding RNAs
Where is expression shown in the Pax6 transgenic mouse?
DNA region upstream of exon confers expression only in the Pancreas and lens pit for exon 0 -other enhancer regions confer expression in specific tissues -Pax gene also displays alternative transcription start sites -all other tissues besides the ones detected dont have the transcription factors needed to transcribe gene.
control elements are
DNA sequence, NOT a protein
Transcription control regions in mouse Pax 6 gene
Different segments of DNA form the Pax6 gene fused to the B-galactosidase reporter gene then the constructs used to make transgenic mice. The constructs then are incorporated into the DNA of all cells in the resulting transgenic mouse embryos but the reporter is expressed in different tissues dependig on which conrtol region is linked to it
Promoter-Proximal elements regulate gene transcription by Pol
II
General transcription factors are required at essentially all promoters utilized by
Pol II
RNA pol 1
RNA transcribed: Pre r RNA function in ribosome components, protein synthesis
RNA pol II
RNA transcribed: mRNA, snRNA, siRNAs, miRNAs, RNA function: encodes protein, RNA splicing, Chromatin mediated respression, translation control
RNA pol III
RNA transcribed: tRNAA, 5S rRNA, snRNA U6, 7S RNA RNA function: protein synthesis, ribosome component, protein synthesis, RNA spiling, Signal recognition particle for inseriton of polupeptides into the ER
Homeodomain transcription factors
SNA binding domain is ~60 amino acids long and is similar to helix-turn-helix motif of bacterial repressors. homeodomains are most often found in transcription factors that function during development and were first discovered in drospholia ex. Ubx
pol II must first bind to
TFIIF before it can join the TBP/TFIIB complex bound to the promoter
Yeast GAL4 transcription factor binds to
UAS control element found in genes required for metabolizing galactose
Deletion constructs of the GAL4 protein can be assayed for binding to
UASgal (using EMSA) and for stimulation of a reporter gene introduced into yeast cells
All nuclear receptor share
a common domain structure Variable region (100-500aa) DNA binding domain (68 aa): two copies of C4 zinc finger motif Ligand bind domain (225-285 aa): ligand binding domain may function as a repressor in some receptors
estrogen binding causes
a conformational change that promotes binding to a co-activator protein that in turn activates pol II.
prior incubation with TBP results in
a footprint, protected region, spanning the TATA box region
testing transcription factor activity by transfection, what is in the first plasmid
a gene encoding a putative transcription factor (protein X) is cloned into an expression plasmid (1)
transcription of the lac operon is prevented by binding of
a tetramer of the lac repressor protein to the operator region
Basic leucine Zipper (bZIP) protein
a-helices in leucine zipper proteins have basic residues at one end and these bind to specific sequences in adjacent major grooves like a pair of scissors.
many activation domains have a high content of
acidic amino acids (glu, asp) or are glutamine rich or proline rich
CAP control
activator that can control transcription of several genes
control of elongation is mediated by formation of
alternative base-paired structures within the first 140nt of the transcript
protein-protein interactions
are often involved in binding multiple transcription factors on adjacent binding sites. -if one factor is bound the second one can bind with greater affinity necuase specific interactions occur with both DNA and the first factor (cooperative binding)
kinds and amounts of proteins produced in a particular cell type
are regulated, unique proteins for different cell types
eukaryotic transcription factors
are the regulatory proteins that bind to specific DNA control elements. humans encode over 2000 transcription factors (yeast several hundred)
Basic-helix-loop-helix (bHLH) protein
bHLH proteins use the same principles but the DNA-binding helices are separated form the leucine zipper by non-helical loops
phosphorylation of pol II CTD is required
before elongation can start
elongation proceeds following
binding of another factor (DSIF) and specific phosphorylation of the pol II CTD domain as well as NELF and DSIF
cAMP-CAP complex
binds to a site just upstream of the lac promoter (interacts with polymerase causing more frequent transcription)
NtrC
binds to a so called enhancer sequence far upstream of the promoter (-108 to -140) -looping of the DNA allows NtrC to bind to sigma 54-pol and activate transcription -requires phosphorylation by a protein kinase called NtrB to activate transcription
effector domain
binds to a specific promoter to activate transcription
combinatorial regulation by bZIP and bHLH proteins
both recognize the same DNA binding site and can bind as either homodimer or heterodimers -in some cases even bZIP and bHLH proteins that recognize different but related DNA binding sites can still bind as heterodimers
the ability to transcribe a gene is controlled at the
chromatin level (closed vs open chromatin) and also at the promoter level (DNA binding transcription factors and other protein complexes assembled near gene promoters)
process of testing transcription factor activity by transfection
co-transfection of the two plasmids in the cells lacking X protein increases reporter gene expression if the transcription factor is an activator or decreases expression if it is a repressor -tells you if protein encoding is in fact binding to the site and affecting transcription
TATA box
commonly found in highly transcribed genes the TATA box is 25 to 35 bp upstream of the start site -consensus sequence 8nt long that is A/T rich
small molecule ligands
commonly regulates bacterial gene transcriptionby altering DNA binding properties of repressor or activator proteins
transcription factor domains function independently
contain only one DNA binding domain-but may contain one or more transcription activation domains
mutations within transcription factor repressor domains often lead to
contitutive (unregulated) expression of the genes they control
the most important mechanism determining differential production
control of transcription initiation and elongation
regulation of elongation provides an additional mechanism for
controlling gene transcription in addition to transcription initiation
footprinting assay incubation prior to DNase I
digestion results in a array of bands reflecting random cutting
most bacterial repressors and many euk transcription factors bind as
dimers and commonly contain a DNA-binding domain called Helix-turn-Helix motif
Pol I and Pol III
each have their own set of general transcription factors that recognize different control elements. since they transcribe only a few genes the control elements are simpler transcription rates by pol I and pol III are tightly coupled to cell growth and poliferation
alternate sigma factors are often used for gene activation in response to
environmental signals
lipid soluble molecules
extracellular signals that diffuse through cellular membranes and directly activate transcription factors ex. cortisol, retinoic acid, thyroxine
order of domains differs depending on
factor, but domains work independently (e.g. activation domain of GAL4 stimulates transcription when used to replace activation domain GCN4
all three eukaryotic polymerases have
five core subunits homologous to the bacterial counterparts
why is the spacing between two promoter elements important?
for recognition and binding by RNA polymerase
who discovered gene regulation in e coli?
fracois jacob and jacques monod, work on lac operon
Enhancers
generally 50-200 bp and comprise multiple individual control elements each 6-10 bp long enhancer sequence binds to multiple transcription factors. -stimulate transcription of any gene even when present thousands of bp away from the start site -you can take enhancer and put by any gene of choice
TFIIH
has helicase activity and melts DNA duplex at start site
silencing gene transcription by heterochromatin formation
heterochromatin reflects a high level of chromatin condensation and is readily observed in telomere and centromere regions where there is no gene transcription
for VDRE (vit D3 response element) TR (thyroid hormone response element) and RARE (retinoic acid response element)
heterodimer nuclear receptors are found exclusively in the nucleus and repress the genes they bind to in the absence of their hormone ligand.
some activation domains are
highly structured, but require binding to a small ligand to change to an active conformation
binding of a nutrient to the sensor protein activates a
histidine kinase activity that in turn activates the response protein (both involve conformational changes)
chromatin condensation is promoted by
histone deacetylation and histone methylation
epigenetic control involves
histone modifications as well as DNA methylation (5-methyl cytosine, often critical in CpG islands) that can trigger chromatin condensation
three structural types of DNA binding domains
homeodomain (homeobox), zinc finger, and leucine zipper
linker-scanning mutation analysis
identifies transcription control elements in promoter regions -mutant constructs are engineered to contain overlapping blocks of scrambled sequence that replace original sequence -promoter region from any gene is linked to a reporter gene for analysis
repressor domains
in place of activation domain that inhibit rather than activate gene transcription. work independently, can be linked to DNA binding domains of any transcription factors
activations domins differ
in their transcription-stimulating potential and are themselves subject to regulation, different combinations of heterodimers can thus elicit varied responses.
epigenetic regulation of transcription
inherited changes in the phenotype of a cell that do not result from changes in DNA sequence -changes in the gene expression initiated by transcription factors are often reinforced and maintained over multiple cell divisions
activation domains function by
interacting with pol II initiation complex
CpG islands
involving genes that are not transcribed at extremely high level, like those containing a tata box or an initiator sequence. -CGCGCG repeated sequences that are not interrupted by anything else. RNA pol recognizes many alternative sites in both directions
in the two component system the phosphate on the his residue
is quickly transferred to a specific Asp residue in the response regulator
CTD phosphorylation
is required for transcription by Pol II and takes place at initiation; phosphorylated CTD also function as a binding domain for mRNA processing enzymes
in the presence of lactose and glucose
lac operon transcription remains low
aniridia
lack of irises in both eyes caused by one defective Pax6 gene (heterozygous)
when lactose is present
lactose binds to the lac repressor protein causing a conformational change and dissociation for the operator
Mediator
large multi-subunit complex that forms bridge between activator domains of transcription factors and pol II -some are required for transcriptional control of all genes (e.g. subunits that bind to pol II and/or maintain overall structure) -other subunits bind to specific activation or repressor domains of transcription factors and are thus required only for activation of some genes
promoter regions
lie upstream of gene transcription start sites and serve binding sites for RNA polymerase and regulatory proteins, required for gene transcription no promoter=no transcription -can be attached to any gene and will govern the transcription of that gene
Yeast RNA pol
looks like bacterial overall but has far more subunits -RPB1 RBP2 similar to the beta and beta prime in bacteria (homologous/evolutionarily related)
Leucine Zipper DNA-binding domains in transcription factors
many proteins including transcription factors contain alpha helices with the hydrophobic amino acid Leu at every seventh position and form coiled-coil dimers -since a-helices have ~3.6 residues per turn, leu at every position makes one side of each helix hydrophobic (amphipathic helix) facilitating their oil around each other to form a dimer (like a zipper)
transcription factors have seperate functional domains due to
modular organization. -transcription-activating function and DNA binding activity reside in different locations (domains) of the protein.
Yeast regulatory transcription control elements
most contain a TATA box that position further upstream compared to higher eukaryotes (90 bp instead of -25 to -35) -most contain only one upstream DNA control element called an Upstream activating sequence (UAS)
promoter elements in e coli
most e coli genes contain two promoter elemetns consisting of specific consensus sequences at -35 and -10 bases upstream of the start site
general transcription factors are all
multimeric proteins
transcription of a single gene is regulated by
multiple DNA control elements and multiple transcription factors -the presence of specific transcription factos that bind to specific control elements differs in different cell types
model of DNA-bound Activators interacting with mediator complex
multiple transcription factors bound to promoter-proximal elements and enhancers make contact with one or more subunits of the mediator complex (require DNA bending) -TAF subunits of TFIID also make contacts with the mediator -magnitude of transcription of a given gene reflects the combined effects of all the contacts (in the absence of a mediator, only minimal basal transcription occurs)
B-interferon gene
multiple transcription factors participate in the cooperative binding of the enhancer sequence. considered an enhanceosome because of the large number of proteins forming complex
NELF
negative elongation factor, pol II pauses after transcribing ~20-50 nts due to binding of this protein complex
When lactose is absent and glucose is present....
no need for the cell to metabolize lactose
Transcription of ribosomal RNA precursor by Pol I
non transcribed spacer region between unites includes a set of enhancer sequences and varying termination sites. pRNA start is involved in silencing transcription SL1 contains TBP and four TBP associated units general transcription factors are bound to promoter (assembly) Pol I assembled at start site
what kind of interactions are between specific amino acids and specific DNA bases?
noncovalent interactions in both euk and prok
phosphorylation
of repressors or activators modulates their DNA binding properties
in general, each transcription factor binds to only
one specific control element
what is required to initiate assembly on TATA box-containing promoters in vitro
only the TBP subunit of TFIID
While testing transcription factor activity by transfection, what is in the second plasmid?
plasmid 2 encodes a reporter gene with a minimal promoter (eg. TATA box only) and an upstream control element for the transcription factor (X binding site)
purpose of the general transcription factors
position the polymerase at the start site and assist in initiation
repressors
prevent pol from binding or from functioning
further reduction in expression of the tryp operon proteins is acheived by
preventing elongation of the transcript (called attenuation) (terminate not to waste any time to transcribe the whole gene) -occurs in bacteria like in eukaryotes
tryp operon
prime example of transcriptional regulation of elongation in bacteria
transcription control elements in genes transcribed by Pol III
promoter elements for tRNA and 5S rRNA genes are uniquely located entirely within the transcribed sequence -U6 snRNA gene however is more like Pol II genes with a TATA box and one promoter proximal element (PSE) -5S and tRNAs primary transcripts are also processed by specialized nucleases
the glutamine synthetase gene A
promoter is recognized by sigma 54-pol
initiator sequences
promoters that contain an initiator sequence that overlaps the start site -initiator sequences are poorly conserved compared to TATA box
transcription factors
proteins that recognize specific DNA sequences
promoter
referes to those DNA sequence elements that lie close tothe start site and recruit as well as position RNA polymerase for transcription initiation
Pax6 gene in humans
regulates a number of developmental pathways and only some mutations lead to aniridia, plays a role in the developmental tissue in the eye
DNA binding domains can be grouped into
related structures with similar motifs
histone deacetylation
removes negative charges and promotes histone-histone interactions (favors condensation)
transcription factors
repressors and activators of transcription that binf to DNA control elements in eukaryotes
DNA-binding sites (control elements) for nuclear receptors are referred to as
response elements
distinguishing features of DNA response elements for nuclear receptors
response elements either contain indirect repeats or direct repeats and bind dimers of nuclear receptor proteins.
sigma protein factor
responsible for recognizing -10 and -35 promoter elements and must first associate with RNA polymerase before it can bind
transcription level for each gene
results from the combined effects of multiple transcription factors bound to its control elements
Prior incubation with TFIID
results in a footprint that extends to about +20 from the transcription start site TSS.
responses to changes in nutrients sometimes involve a
sensor protein that does not bind to promoters and a response regulator protein that does
CTD
seven amino acid repeat (26 in yeast, 52 in vertebrates
E coli has a total of
seven sigma factors each recognizing a specific promoter consensus sequence
what does analysis of control regions using a single bp show?
shows that the critical elements consists of a specific sequence 6-10 bp long -these short sequences serve as a binding site for specific transcription factors.
DNA control elements
span a larger region of DNA than in bacteria, recruit RNA polymerase aside from promoter elements
What determines binding affinity of DNA binding protein
specific contacts between atoms in the recognition alpha helix (sequence reading helix) and the major groove of DNA
genes that require alternate sigma factors generally control a
specific function
TBP
subunit of TFIIIB, TBP is thus required by all three RNA polymerases
when glucose falls how does the cell respond
synthesizes cyclic AMP (cAMP) which binds to a dimer of the catabolite activator protein (CAP)
example of a ligand binding to activation domain
tamoxifen used for breast cancer blocks binding co-factor -normal ligand estrogen is the agonist and inhibitors of the binding is the antogonist
RNA Pol II is the only pol that has
the c-terminal domain (CTD) in its largest subunit, RNA pol copies mRNA.
For GR (glucocorticoid response element) and ER (estrogen response element) ligand binding occurs in
the cytoplasm and promotes translocation of the receptor to the nucleus
what governs the transcriptional level of a gene
the promoter region and its response to regulatory signals
most e coli gene transcription require
the sigma 70 factor
when tryptophan is available as a nutrient...
the trp repressor protein reduces transcription of the operon
nucleosomes in decondensed chromatin generally do not prevent binding of general or specific transcription factors becuase
they can "slide" along the DNA
Herpes Virus thymidine kinase gene contains how many control elements
three control elements assayed by transfection
control of transcription involves
three distinct DNA elements: CAP site, promoter, and operator
lac operon encodes for
three proteins that metabolize lactose (lacZ encodes B-galactosidase the first gene in the operon)
Human Pax6 Gene reveals
tissue-specific expression
enhancers are often responsible for
tissue-specific transcription of gene. you can put an enhancer next to any gene but if tissue does not contain the right transcription factors than it will have no effect
why do cells need to regulate gene expression
to respond to or adapt to changes in its environment (prokaryotes and eukaryotes) and to generate different types of cells during development of multicellular organisms (differentiation)
estrogen receptor
transcription factor with an activation domain that binds to estrogen (small lipid soluble hormone)
lipid soluble molecules bind directly to
transcription factors called nuclear receptors present either in the cytoplasm or the nucleus
reaction cascade is triggered by
transmembrane receptors in response to extracellular signals, reaction cascade leads to activation or repression of a transcription factor already present in the cell often involving phosphorylation
Bacterial RNA pol
two alpha subunits, omega subunit, sigma subunit to recognize promoters, beta and beta prime sububit. similar shape to the eukaryotic yeast RNA pol
some activation domains, especially acidic ones remain
unstructured, do not fold into a specific tertiary structure until they bind to a co-activator protein
enhancers can be located
upstream, downstream within introns, or after the last exon
column chromatography
used to separate proteins from cell extracts into different fraction based on charge size or hydrophobicity. -column fractions are then incubated with a radiolabeled DNA fragment containing a control element (probe) followed by gel electrophoresis -when a transcription factor binds to the control element, a slower migrating protein-DNA complex is observed
electrophoretic mobility shift assay (EMSA)
useful method for detecting transcription factors during purification
DNA-binding domain determines
which genes can be stimulated for transcription but activation domains work when attached to any transcription factor DNA-binding domain
why use linker-scanning mutation analysis?
you can take constructs you put into the cell, if scrambled sequence with no mRNA made then it must mean that there is a control sequence in that region that controls transcription of that gene. if there is no effect on RNA synthesis then its not important to the control region. end up with a map of where the control elements are.
zinc finger DNA binding domains in transcription factors
zinc finger motifs are common in transcription factors (sometimes in multiple copies) but are also found in other cellular proteins. -cysteines and histidines in the motifs bind zinc ions via their sulphydryl groups
