Biology 340 Molecular Biology
Lectures 19, 20 & 21
Regulation of Gene Expression in Eukaryotes
Mar. 12, 14, 16, 2001
Reading: Chap. 10 Lodish et al. pp. 358-400
Outline:
1. Eukaryotic transcription
2. Regulatory elements that control transcription
3. Transcription factors
a. DNA binding domains
b. activation domains
4. RNA polymerase II
5. Steroid hormone regulation
Lecture:
1. Eukaryotic transcription
Eukaryotes:
--Most are multicellular and made of different cell types.
--Different cells express distinct subsets of genes.
--Gene expression is regulated so genes are turned on when they are needed
during development and in the correct cell types.
--Most genes in higher eukaryotes are regulated by controlling their transcription.
General principles:
1. Transcription begins at a specific site or a cluster of neighboring sites.
(Mapped using primer extension or nuclease protection)
2. Transcription is controlled by many regulatory proteins that interact with DNA
sequence elements or with other proteins or RNA polymerase.
3. The "promoter" of a eukaryotic gene is less defined than a prokaryotic
promoter; it generally consists of many sequence elements located upstream of
the transcription unit.
Eukaryotic RNA polymerases:
1. RNA polymerase I transcribes precursor rRNA, which is processed to give
28S, 5.8S, and 18S rRNAs.
2. RNA polymerase II transcribes all protein-coding genes into pre-mRNAs.
Transcription by "Pol II" is sensitive to the drug -amanitin.
3. RNA polymerase III transcribes genes for tRNAs, 5S rRNA and other small
RNAs involved in RNA splicing.
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All contain 3 large subunits (like the , ' subunits of bacterial RNA polymerase)
and 12-15 smaller subunits.
Genetic analysis to identify regions of a gene essential for expression:
See Figure 10-24
1. Isolate the region of a gene thought to contain the 5' end and the sequences
upstream.
2. Construct mutant vectors containing different portions of the 5' end fused to a
"reporter gene"--a gene for an easily assayed enzyme such as luciferase or
chloramphenicol transferase.
3. Introduce the vectors into separate plates of mammalian cells by transfection.
4. Measure the activity of the reporter enzymes. The greater the activity, the
better the regulatory region activates gene expression.
2. Regulatory elements that control transcription
Analysis of mutants that contain different deletions or point mutations
(scanning linker mutagenesis) in the putative regulatory region has defined a
number of conserved regulatory elements that control transcription.
a. TATA box
b. Sp1 site
-35 to -25
up to -100
TATAA/TA
GGGCGG
Other regulatory elements are found in genes expressed only in certain tissues
or that are coordinately controlled in response to a particular cellular signal.
Globin genes: CCAAT box
Muscle specific genes: E box, CANNTG
--Elements such as the TATA box and Sp1 sites are found in close proximity to
the start site of the gene and are often referred to as proximal promoter
elements.
--Some regulatory elements are found at thousands of base pairs upstream of
the gene, within introns, or even downstream. These are known as enhancers;
many can function in either orientation.
--Most eukaryotic genes are regulated by multiple transcriptional control
elements. See Fig. 10-34 for hypothetical mammalian gene. These elements
serve as binding sites for a variety of DNA binding regulatory proteins, which
each bind specific regulatory elements.
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3. Transcription factors
--Transcription factors are specific regulatory proteins that either bind DNA or
bind to RNA polymerase or do both to stimulate (or repress transcription).
--Identify higher eukaryotic transcription factors by biochemical analysis:
DNase I footprinting, gel mobility shift assays.
--Transcription factors are modular proteins made of distinct functional domains,
including DNA binding and activation domains.
--Genetic analysis can be used to identify domains. See Fig. 10-38 for genetic
dissection of the yeast Gal4 protein, a transcription activator that stimulates
transcription of yeast genes required for growth on galactose.
DNA binding domain: amino terminus
Activation domain: carboxy terminus
a. DNA binding domains
--Many transcription factors contain similar structural domains that bind DNA.
i) homeodomain Drosophila antennapedia, human HOX genes
ii) zinc finger
cys4 type Steroid hormone receptor superfamily
cys2his2 type TFIIIA (RNA polymerase III transcription factor)
iii) leucine zipper GCN4 (yeast regulator of amino acid metabolism)
iv) helix-loop-helix (HLH) MyoD
b. activation domains
--these are the regions of the proteins that bind other proteins, such as subunits
of RNA polymerase, to stimulate transcription.
--Less easily defined based on structural motifs, more often identified genetically.
-- acidic activation domains are somewhat common.
4. RNA polymerase II complex
a. RNA polymerase II is assembled from subunits on the DNA to initiate
transcription. See Fig. 10-50 for the in vitro process
b. TBP (TATA box binding protein, a subunit of a general transcription factor,
TFIID) binds to DNA at the TATA box.
c. TFIIF and RNA Polymerase II bind to TFIID on DNA to form a preinitiation
complex.
d. TFIIE and TFIIH are added to form the transcription-initation complex.
e. Nucleotide triphosphates serve as substrates for transcription.
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f. Most of the general transcription factors are released as the RNA polymerase
II carries out transcription.
5. Model for the role of transcription factors in activating gene expression
See CD-ROM animation "combinatorial control of transcription" and Fig. 10-61
--Transcriptional activators stimulate the assembly of initiation complexes.
--Proteins bind to proximal promoter elements and enhancer elements.
--Other proteins called coactivators bind to the transcription factors on the DNA.
--The coactivators contact RNA polymerase II and help stabilize the initiation
complex assembled on the TATA box.
--The more rapidly and stably the initiation complex is formed on DNA, the more
a gene is transcribed.
--different levels of gene expression are possible because not all regulatory
elements bind proteins at any one time.
--Certain combinations of factors have a greater effect on transcription initiation.
6. Steroid hormone regulation
--Steroid hormones include a number of important physiological regulators
estrogen, progesterone: regulate female reproductive system
androgens, such as testosterone: regulate male reproductive system
--The lipid-soluble steroids, retinoids, vitamin D, and thyroid hormones bind to
nuclear receptors that activate transcription of particular subsets of genes.
--The nuclear receptors share similar functional domains (Fig. 10-64)
amino terminal domain (variable)--DNA binding domain--carboxy terminal
hormone binding/activation domain
--The DNA binding domain consists of cys4 zinc fingers.
--The DNA sequence elements bound by these nuclear receptors are similar and
consist of 6 bp inverted repeats or direct repeats with 3-5 nts between repeats
(Fig. 10-65).
--The receptors bind DNA as dimers.
Sequence of events involved in hormone signaling:
See Fig. 10-67 for signaling by the steroid hormone, cortisol.
a. In the absence of hormone, the receptor is sequestered in a complex with
Hsp90 (a heat shock protein).
b. Hormone diffuses across the lipid bilayer.
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c. Hormone binds to the receptor, changes its conformation and releases it from
the inhibitor, Hsp90.
d. The hormone-receptor complex moves to the nucleus.
e. The receptor binds to DNA at the response element and activates
transcription of the target genes.
f. The proteins produced from the target genes carry out the necessary cell
response to the hormone.
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