BI 213 Exam: Chapter 9.2: Transcription Factors in Eukaryotes
What are the two transcriptional regulatory proteins; and how do they usually regulate
Activators and Repressors by recruit histone modifying enzymes and nucleosome remodeling factors
How many sequence elements does an enhancer have?
An important aspect of enhancers is that they usually contain multiple sequence elements that bind different transcriptional regulatory proteins. o These proteins work together to regulate gene expression.
What is CTCF?
An insulator Specific sequences bound by specific DNA-binding proteins: CTCF CTCF binds to insulator sequences, which are nearby/flanking chromosomal domain TAD
What are transcriptional regulatory proteins
Broader term for TF's (don't all necessarily bind DNA; TF's directly contact DNA)
Key notion: DNA looping allows transcription factors at enhancers to interact with proteins at promoters.
Key notion: DNA looping allows transcription factors at enhancers to interact with proteins at promoters.
Key notion: Enhancers contain binding sites for multiple transcription factors and regulate most tissue-specific and inducible genes.
Key notion: Enhancers contain binding sites for multiple transcription factors and regulate most tissue-specific and inducible genes.
How are eukaryotic genes regulated? (key notions)
Key notion: Eukaryotic genes are regulated by proteins that bind to cis-acting sequences.
Key notion: Regulatory sequences can be located at distant enhancers as well as near promoters
Key notion: Regulatory sequences can be located at distant enhancers as well as near promoters
Key notion: Transcription factor binding sites in cells can be identified by chromatin immunoprecipitation.
Key notion: Transcription factor binding sites in cells can be identified by chromatin immunoprecipitation.
Which main proteins are responsbile for DNA looping
Mediator and Cohesin Mediator and Cohesin associated with enhancers/promoters are responsible for DNA looping
How many sequence elements does an enhancer have? How does this make enhancers specific?
Multiple binding sites for different types of TF This combination is specific Can have a range of individual binding sites for TF in an enhancer This combination makes it specific Many gene-specific TF bind to one enhancer. All have to bind and be present to allow enhancer to function
Naya's luciferase reporter assay
Naya identified a novel muscle gene used luciferase reporter gene to understand its expression bar on the left (control group without regularoy sequence) bar on the right: set of cells TRANSFECTED with regulatroy seqeuence (ccandidate sequence of the muscle gene) - transfect cells and extract lysate to put into machine since bar on the right (independent varaible) showed more luciferate units (more light emitted); it confirms the increased transcription of reporter gene, and thus the identifying the gene regulatory sequence.
What does the DNA looping tell us about the mediator protein?
Not only is the Medaitor important in preinitiation complex and its itneratction with gene specific TF It also works together with cohesin hulahoop protein to induce DNA looping to bring gene specific enhancer to TSS regulation
does the luciferase assay express cDNA
Not overexpressing a cDNA. Rather, you put your reg. sequence upstream of a reporter cDNA, i.e. Firefly luciferase If it really is a regulatory sequence, the cDNA will be transcribed
What are cis-acting regulatory sequences
Promoters and enancers the binding of proteins to cis-acting sequences (e.g., the lac operator) that control the transcription of adjacent genes.
Gene regulation focus
Promotor is very clos eto transcription start site (that is what a promoter is)
How is protein binding detected in EMSA
Protein binding is detected as a decrease in the electrophoretic mobility of the DNA fragment, since its migration through the gel is slowed by the bound protein.
How many genes can be in one DNA loop designed for enhancer specificity?
Some TF can regulate more than one type of gene In a DNA loop, there can be many other genes Compartmentalize genes that share transcription factors Concentrate them into one area - chromosomal domain of TAD
1. How do enhancers differ from promoters as cis-acting regulatory sequences in eukaryotes?
The activity of enhancers depends on neither their distance nor their orientation with respect to the transcription start site. Promoters are defined as being near the transcription start site.
What can you combine EMSA with to lead to the characterization of transcription factor binding sites within enhancers and promoters of eukaryotic
The combined use of electrophoretic-mobility shift assays and detailed mapping of protein-DNA interactions
After you have isolated the fragment of DNA linked to the TF OF INTEREST using immunopericiation and antibody, what do you do?
The formaldehyde cross-links are then reversed, and the immunoprecipitated DNA is isolated and analyzed to determine the sites (DNA SEQUENCE) to which the specific transcription factor was bound within the cell.
Give an example of how an enhancer has multiple sequence elements that bind different transcriptional regulatory proteins.
The immunoglobulin enhancer spans 200 bp contains at least nine distinct sequence elements that serve as protein binding sites for different TF The overall activity of the enhancer reflects the combined action of the proteins associated with each of its individual sequence elements
Important note about enhancers
The overall activity of the enhancer reflects the combined action of the proteins associated with each of its individual sequence elements
You are studying the enhancer of a gene that normally is expressed only in neurons. Constructs in which this enhancer is linked to a reporter gene are expressed in neuronal cells but not in fibroblasts. However, if you mutate a specific sequence element within the enhancer, you find expression in both fibroblasts and neuronal cells. What type of regulatory protein do you expect binds the sequence element?
The sequence would be the binding site of a re-pressor that prevents transcription in fibroblasts.
3. How do DNA loops generated by CTCF affect the action of enhancers
They allow enhancers to interact with promoters within a looped domain, but prevent enhancers from interacting with promoters in a different domain
How can enhancers function by binding transcription factors that then regulate RNA polymerase, when enhancers are separated by long distances from TSS
This is possible because of DNA looping, which allows a transcription factor bound to a distant enhancer to interact with proteins associated with Mediator or general transcription factors (e.g., TFIID) at the promoter
What are topologically associated domains?
Topology: the way in which constituent parts are interrelated or arranged. A topologically associating domain is a self-interacting genomic region, meaning that DNA sequences within a TAD physically interact with each other more frequently than with sequences outside the TAD.
• Figure 9.8 DNA looping
Transcription factors bound at a distant enhancer are able to interact with Mediator or general transcription factors at the promoter because the intervening DNA can form loops, stabilized by cohesin.
What is the significant implication of transcription factors?
Transcription factors bound to distant enhancers can thus work by the same mechanisms as those bound adjacent to promoters, so there is no fundamental difference between the actions of enhancers and those of cis-acting regulatory sequences adjacent to transcription start sites
How does luciferase indicate amount of gene transcribed and expressed
Unit: luciferase- how much light is emitted and detected
Application of the multiple sequence elements (protein binding site for different TF) in an enhancer
Using reporter assay (luciferase) determine the relative importance of each binding site of gene-specific TF See how lucierafase actitiy changes (increased or reduced) if you delete or mutate a binding site such as E or π i n immunoglobin enhancer Define the sequences
after isolating a specific TF binding site; how can you test the binding of a transcription factor to a specific regulatory element
Want to know whether original sequence is from myosin? Use PCR and amplify obintaed DNA sequence, using primers for mysosin Note: If you want to know, "is this particular gene regulated by this TF?" just PCR for that target sequence from all the fragments. Can also be a control to confirm the right TF is being assayed ("I know this TF regulates myosin"..."Oh, it also regulates these other 99 genes")
When is the use of a reporter gene of Firefly luciferase
We have identified a sequence that we SUSPECT is a regulatry sequence to find out whether this gene is THE PROMOTER or NEAR the promoter UPSTREAM, and whether is it ACTIVE Do molecular cloning of the identified gene with reporter gene luciferase Use PCR and restriction enzyme to clone to plasmid intorduce recombinant plasmid into cells in culture (transfection) and assay using reporter gene
How are enhancers specific?
Where enhancer located and what TF's bind to it determines its gene specificity
Are repressors as common as activators in eukaryotic cells?
Yes sir
7. How could you experimentally distinguish between a gene regulated at the level of transcription initiation and one regulated at the level of elongation?
You could use chromatin immunoprecipitation with antibodies against the factors that inhibit elongation, NELF and DSIF, to determine whether they are bound to RNA polymerase near the start site of your gene. In addition, you could use an antibody against phosphorylated serine 5 of the polymerase CTD to determine if transcription has initiated but the polymerase has paused near the start site (elongation).
You have identified a sequence ACGTTG that, when mutated, abolishes the function of an enhancer in gene transfer assays. How could you identify and purify a transcription factor that binds to this sequence?
You could use electrophoretic mobility shift assays to determine that cell extracts contain a pro-tein that binds to oligonucleotides containing the sequence ACGTTG. You could then use affinity chromatography to purify the protein.
6. You have identified a novel transcription factor called TXF, which you think is involved in the differentiation of different types of muscle cells. How could you identify the sites on DNA to which TXF binds in heart cells compared to skeletal muscle cells?
You would want to use chromatin immunopre-cipitation of TXF, followed by large-scale DNA sequencing to identify binding sites in both cell types (CHIP-seq)
Describe electrophoretic-mobility shift assay (EMSA)
a radiolabeled DNA fragment is incubated with a protein preparation and then subjected to electrophoresis through a nondenaturing gel
What is the biggest difference between enhancers and promoters?
an enhancer is typically further upstream of the promoter Enhancer: can either be upstream or downstream from start site Enhancer can form a loop at the end and come back to DNA sequence without interfering Promoter must be fixed in one orientation Enhancer is more dynamic (can be flipped in either oritentation) and still stimulates transcription
How can transcriptional repressors indirectly inhibit transcription?
many repressors (called active repressors) contain specific functional domains that inhibit transcription via protein-protein interactions Repressors can inhibit transcription by interacting with specific activator proteins, with Mediator proteins or general transcription factors, and with corepressors that act by modifying chromatin structure.
after isolating a specific TF binding site; how can you test the binding of a transcription factor to a specific regulatory element
using PCR to detect the sequence of interest in chromatin immunoprecipitates
How can TF at enhancers interact with proteins at promoters
via DNA looping
How are chroma-in extracted from cell fragmented in chromatin immunopercipitation? how can the fragment of DNA linked to the TF OF INTEREST be isolated?
via sonication fragment of interest can be isolated with an antibody against the transcription factor
Why is gel electrophoresis used in EMSA?
visualization
Figure 9.7 Action of enhancers
• Without an enhancer, the gene is tran-scribed at a low basal level (A) • . Addition of an enhancer, E—for example, the SV40 72-base-pair repeats—stimulates transcription. • The enhancer is active not only when placed just upstream of the promoter (B) but also when inserted up to several kilobases upstream or down-stream from the transcription start site (C and D). • In addition, enhancers are active in either the forward or backward orientation (E)
GFP vs luciferease
**Luciferase assay is preferred b/c it is chemical reaction that yields quantifiable results (vs GFP would just be fluorescence, saturation)
After isolating a specific TF; how can you identify all the other sites that TF binds to WITHIN A CELL?
, a genome-wide analysis of the immunoprecipitated DNA fragments, either by large-scale DNA sequencing or by hybridization to microarrays
Sumarize Chromatin immunocpercipatiton
-Culture cells. Then at specific time, fix cells, aka "cross link" w/ formaldehyde. DNA and protein that were associated together stay fixed together -Extract chromatin, fragment it (sonication) -Incubate fragmented chromatin w/ Ab against target TF -Wherever TF is bound, Ab will precipitate those chromatin fragments (could be thousands of fragments) -Reverse cross-links, get ride of Ab/protein -Purify DNA fragments. Sequence -Hopefully, sequences obtained are (very) similar to each other
How far can enhancers be from promoters in mammalian cells?
50 kilobases away Enhancers can be more than 50 kb away from promoters in mammalian cells
How can you differentiate a promoter from an enhancer
: clone each in a reporter gene Put promoter way upstream- wont transcribe Put promoter way downstream- wont trasncribe Enhancer way upstream or downstream- transcribe
How can transcriptional repressors regulate transcription?
1) bind directly to SPECIFIC DNA sequences and inhibit transcription (compete directly with activators) 2) or INDIRECTLY by interfering with the binding of other transcription factors to DNA via protein-protein intreactions
Summarize the action of eukaryotic transcriptional repressors
1. Bind to DNA site: prevent activtor from binding 2. Actively repress: inhibit transcription through direct interaction (Also recruit co-repressors)
What is the activation domain of transcriptional activators?
1. specific amino acid sequences that help stimulate transcription
Explain topologically associated domains (TADS) and how they maintain chromatin specificity?
Compartmentalize genes that share transcription factors Concentrate them into one area - chromosomal domain of TAD CTCF insulator and overall DNA loop p prevent crossover interactions between enhancers outside and inside domain
What are cis-acting regulatory sequences?
Control the transcription of adjacent genes - in prokaryotes e.g. lac operator in eukaryotes promoter → bind general TFs enhancer → bind gene-specific TFs
What are the two INDEPENDENT domains of transcriptional activators?
DNA-binding domain activation domain: stimulates transcription by interacting with other proteins
What are the domains of transcriptional repressors?
DNA-binding sequence many have functional domains that inhibit transcription via protein-protein interactions
Can TF's bind to different binding sites in the genome?
Even though enhancer are gene specific- a TF binding to myosin gene, it may also (TF) bind to a immunoglbin gene (Different gene)
EMSA
EMSA also called "gel shift assay" -Synthesize oligonucleotide (~20 bp) of that predicted sequence. Tag it (often radioactive) -Make protein in vitro, the transcription factor -Incubate the TF with the labelled oligonucleotide -In another tube, incubate oligonucleotide alone -After ~15 min -Run gel electrophoresis
What is the difference between EMSA and Chromatin immunopercipitation
EMSA- one transcription factor and one sequence at a time Chromatin immuno-precipitation: isolates one TF factor and one sequence, but can lead to further chip-sq to determine where the TF is binding in the entire genome. It starts off by isolated one gene regulated by TF (binding site), and you can determine all the other genes that TF binds to using CHIP-SEEK IRANY
What are the sequences that control eukaryotic genes far away from the TSS called?
Enhancers
How do enhancers function similarly to promoters?
Enhancers, like promoters, function by binding transcription factors that then regulate RNA polymerase.
What is another name for EMSA
Gel shift assay
Main takeawy for the ideal eukaryotic promoter of the herpes virus thymidine kinase gene
Have proximal elements (TATAA box, et al.) and neighboring elements (-100 to -50 bp)
Current challenges with transcription factors in eukaryotes
Identification and testing regulatory sequences (Both negative and positive) in eukaryotes
What do you find in TADS
In the DNA loop, there'd likely be many other genes. These genes that are together in the loop are in the topologically associated domain (TAD) They can share some (genera) TF's that bind to the enhancer sequences. Compartmentalize genes with similar TF's within a TAD
How were binding sites of transcriptional regulatory proteins (TF) in promoter or enhancer sequences have been identified
by EMSA electrophoretic-mobility shift assay
How can you identify a specific TF protein binding site?
by EMSA: you have a suspected specific TF binding site DNA sequence clone it or synthesize a double-stranded copy of that sequence ( around 20bp) - tagged with a radioactive probe (this is a binding assay) make the target TF of that specific protein binding site by IN VITRO TRANSLATION two test tube: one: protein (TF) with radiolebellled ds-DNA fragment other one just contains ds-DNA fragment without protein TF (control group) incubate both perform GE to fractionate and visualize the binding One lane: dna-protein complex + free dna other lane: free dna only Free DNA is smaller than dna-protein complex Free DNA is found at the bottom- migrated faster
How can TF binding sites IN CELLS be identified?
by chromatin immunoprecipitation. determining the regions of DNA that bind a transcription factor
How are the DNA loops stabilized?
by cohesin, which forms a ring structure that encircles two strands of DNA. hula hoop cohesin protein
After identifying regulatry regions of eukaryotic genes, how can the role of specific sequences within this region be determined?
by in vitro mutagenesis o active regulatory regions can be identified using reporter gene such as firefly luciferase, and in vitro mutagenesis can be used to determine the roles of specific sequences within the region.
How does the activation domain of transcriptional activators STIMULATE transcription
by interacting with other proteins through TWO DISTINCT MECHANISMS: 1. Intreacts with Mediator and general transcription factors (TFIIB e.g): this is important for stabilizing transcriptional complex (assembly of RNA polymerasE) and STIMULATE TRANSCRIPTION 2. intreact with CO-ACTIVATORS that stimulate transcription by modifying chromatin structure.
How do co-repressors repress transcription?
by modifying chromatin structure similar to co-activators
How do CO-activators stimulate transcription?
by modifying chromatin structure.
How is transcription controlled in eukaryotic cells
by proteins that bind to specific regulatory sequences and modulate the activity of RNA polymerase. these sequences are often located at substantial distances from promoters and multiple transcription factors act in concert to regulate target genes.
Where were the two regulatory sequences that are found in many eukaryotic genes identified?
by studies of the promoter of the herpes simplex virus gene that encodes thymidine kinase (Figure 9.5).
How were eukaryotic regulatory sequences (cis-acting and other) identified?
by the use of gene transfer assays to study the activity of suspected regulatory regions of cloned genes (figure 9,.4)
How are eukaryotic genes controlled by regultaroy sequences?
can be near promoters or can be located FAR away from the transcription start/initaiton site (TSS)
Summarize Figure 9.12 Chromatin immuno-precipitation
grow cells in culture 1. Treat cells with formaledhyde to link DNA (binding site of TF) to protein (TF) 2. sonicate to produce fragments of chromat 2. immunopercipitate with antibody against TF of interest 3. antiboy binds to specific TF and immunopericiates it 4. collect chromatin-antibody complex 5. reverse-cross links 6. purify DNA 7. now you have isolated the DNA fragment containing a specific TF BINDING SITE. you could continue in two ways: more specific or more general specific: test for individual binding sites by PCR general: genome-wide analysis of binding sites by microarrays or high-throughpout cequences (CHIP-SEQ)
What are gene-specific regulatroy factors/TF
control the expression of individual genes.
Why is crosslinking imporant in chromatin immunopercipitation?
cross-linking proteins to DNA in cell o As a result, transcription factors (proteins) are covalently linked to the DNA sequences to which they were bound within the living cell.
What are insulators?
divide chromosomes into independent domains & prevent enhancers from acting on promoters located in an adjacent domain. The main protein that binds insulators in vertebrates is CTCF.
Where were enhancers first identified?
during studies of animal virus, SV40 (Figure 9.6).
What is the biggest difference between enhancers and promoters?
in eukaryotes promoter → bind general TFs enhancer → bind gene-specific TFs
4. Draw a diagram showing how alternative splicing could produce both a repressor and an activator from the same gene.
interesting note: alternative splicing can produce both a repressor and activator FROM SAME GENE
What is transfection
introducing recombinant plasmid into cells on culture
What were the two regulatory sequences that are found in many eukaryotic genes identified in herpes simple virus gene
o Both of these sequences are located within 100 base pairs upstream of the transcription start site: Their consensus sequences are CCAAT and GGGCGG (called a GC box). o Specific proteins that bind to these sequences and stimulate transcription
Describe Figure 9.4 Identification of eukaryotic regulatory sequences
o The eukaryotic regulatory sequences are usually added upstream of a reporter gene that encodes an easily detectable enzyme, such as firefly luciferase (the enzyme responsible for bioluminescence).
in EMSA, why is a non-denaturing gel used?
so that the protein remains bound to DNA. (radiolebelled DNA with probe to target protein binding site of TF)
What to Transcription factors binding to?
to short degenerate DNA sequence · The binding sites of most transcription factors consist of short DNA sequences, typically spanning 6-10 base pairs.
What is an enhancer specific to? How does it maintain this specificity?
the activity of any given enhancer is specific for the promoter of its appropriate target gene. This specificity is maintained by the organization of chromosomes within the nucleus into looped domains of approximately 100 to 1000 kb, formed by the interaction of two molecules of an architectural protein called CTCF and cohesin (Figure 9.10). · Enhanc-ers can interact with promoters within the same looped domain, but are prevented from interacting with promoters in other domains, presumably by the structure of chromosomes within the nucleus.
How can TF binding sites in promoters and enhancers be identified? (which experiment)
the electrophoretic-mobility shift assay EMSA
What is the application of the firefly luciferase
the enzyme responsible for bioluminescence This is an enzyme it's gene is USED AS A REPORTER gene to identify eukaryotic regulatory sequences the eukaryotic regulatroy sequences are added UPSTREAM (after transcritpion intiation site) of the reporter gene so anything transcribed after the reproter gene gives a detectable phenotype provides a sensitive ASSAY for the ability of the cloned regulatory sequences to direct transcription.
How can enhancers stimulate transcription?
their activity depended on neither their distance nor their orientation with respect to the transcription initiation site o They could stimulate transcription when placed either UPSTREAM or DOWNSTREAM of the promoter, in either a forward or backward ORIENTATION
What was surprising by the discovery of enhancers in the SV40 virus gene promoter?
their activity depended on neither their distance nor their orientation with respect to the transcription initiation site (Figure 9.7).
Key Notion: Transcription factors bind to short degenerate DNA sequence. What does degenerate mean?
these binding sites are degenerate, meaning that the transcription factor will bind not only to the consensus sequence but also to sequences that differ from the consensus at one or more positions.
What are the similarities between enhancers and promoters?
they are DNA sequence that are recognize by DNA binding proteins known as transcription factors
What is important to remember about the regulatory sequences in the herpes thymdine kinase promoter
they are located close to the promoter and initation start site relatively simple organization of CCAAT and GC boxes
Summary of genes to be transcribed by RNA pol II
they have core Promoter elements, including TATA box and other sequences which collectively serve as a specific binding site for general transcription factors
Where do the cis-acting regulatory sequences of gene-specific TF located?
though not always, located upstream of the transcription start site. (NOT ALWAYS)
What gives enhancer specificity
where it is placed in relation to gene, and what factors is bound to it
does the luciferase assay express cDNA Not overexpressing a cDNA. Rather, you put your reg. sequence upstream of a reporter cDNA, i.e. Firefly luciferase If it really is a regulatory sequence, the cDNA will be transcribed
you are not expressing a cDNA you are cloning a regulatry sequence upstream of a CDNA copy of a reporter gene of the enzyme firefly luciferse thus, you moelcualr clone the identified gene the cDNA copy is of the REPORTER GENE, not the identified suspected gene Not overexpressing a cDNA. Rather, you put your reg. sequence upstream of a reporter cDNA, i.e. Firefly luciferase If it really is a regulatory sequence, the cDNA will be transcribed
Figure 9.15 Action of eukary-otic repressors
§ (A) Some repressors block the binding of activators to regulatory sequences. § (B) Other repressors have active repression domains that inhibit transcription by interactions with Mediator proteins or general transcription factors, as well as with corepressors that act to modify chromatin structure.
Figure 9.11 Electrophoretic-mobility shift assay
· A sample containing radiolabeled fragments of DNA is divided in two, and half of the sample is incubated with a protein that binds to a specific DNA sequence. · Samples are then analyzed by electrophoresis in a nondenaturing gel so that the protein remains bound to DNA. · Protein binding is detected by the slower migration of DNA-protein complexes compared to that of free DNA. Only a fraction of the DNA in the sample is actually bound to protein, so both DNA-protein complexes and free DNA are detected following incubation of the DNA with protein.
Figure 9.12 Chromatin immuno-precipitation
· Cells are treated with formaldehyde to cross-link DNA and proteins sonication to produce fragments of chromatin . · The chromatin fragments are incubated with an antibody against a specific transcription factor, and chromatin fragments bound to the antibody are collected as de-scribed in Figure 5.11. (immunopercipitation) · The cross-links are then reversed, and DNA is purified to yield DNA fragments containing binding sites for the specific transcription factor of interest. The immunoprecipitated fragments can be analyzed either by PCR to test for the presence of a specific DNA sequence or by global methods, such as hybridization to microarrays or high-throughput DNA sequencing, to identify all of the binding sites for the transcription factor within the genome.
Figure 9.10 Chromosomal domains and CTCF
· Chromatin within the nucleus is organized into looped domains formed by the interaction of CTCF and cohesin. · Enhancers are restricted to interacting with promoters in the same domain.
Figure 9.9 The immunoglobulin enhancer
· The immunoglobulin heavy-chain enhancer spans about 200 bases and contains nine functional sequence elements, which together stimulate transcription in B lymphocytes.
How is the specificity of enhancers maintained?
· The specificity is maintained by the organization of chromosomes within the nucleus into looped domains of approximately 100 to 1000 kb, formed by the interaction of two molecules of an architectural protein called CTCF and cohesin (Figure 9.10). · Enhanc-ers can interact with promoters within the same looped domain, but are prevented from interacting with promoters in other domains, presumably by the structure of chromosomes within the nucleus.
Figure 9.14 Transcriptional activators
· Transcriptional activators consist of two independent domains. · The DNA binding domain recognizes a specific DNA sequence. · The activation domain interacts with Mediator or other components of the transcriptional machinery. In addition, activation domains interact with coactiva-tors that facilitate transcription by modifying chromatin structure
Figure 9.6 The SV40 enhancer
• The SV40 promoter for early gene expression contains a TATA box and six GC boxes arranged in three sets of repeated sequenc-es. • In addition, efficient transcription requires an upstream enhancer consisting of two 72-base-pair (bp) repeats
How are enhancers specific?
• The binding of specific transcriptional regulatory proteins to enhancers is responsible for the control of gene expression during development and differentiation • A well-studied example is the enhancer that controls the transcription of immunoglobulin genes in B lymphocytes. Gene transfer experiments have established that the immunoglobulin enhancer is active in lymphocytes, but not in other types of cells.
Figure 9.5 A eukaryotic promoter
• The promoter of the thymidine kinase gene of herpes simplex virus contains three sequence elements upstream of the TATA box (more negative)that are required for efficient transcription: two GC boxes (consensus sequence GGGCGG) and a CCAAT box Gene regulation focus: Promoter is very close to transcription start site (as it should be)
Figure 9.4 Identification of eukaryotic regulatory sequences
• The regulatory sequence of a cloned eukaryotic gene is ligated to a reporter gene that encodes an easily detectable enzyme. • The resulting plasmid is then introduced into cultured recipient cells by transfection. • An active regulatory sequence directs transcription of the reporter gene, expression of which is then detected in the transfected cells.