| M W F; 10:10 - 11:00 pm |
Molecular Biology |
Douglas W. Smith |
| York 2722 |
BIMM 100 |
5254 Muir Biology Building |
| Fall, 2000 |
x42620; dsmith@ucsd.edu |
| BIMM100 | Syllabus
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Readings: Brown, 8: 188-192; 7: 162-168; 11: 266-272
Outline:
A. Short Sequences (Upstream Elements) WITHIN RNA Pol II Promoters:
1. Sites: ... [Brown, Fig 8.9, 8.12]
Have already seen in discussion of the RNApolII Basal Transcription Apparatus that RNApolII promoters usually contain 2 elements:
1. The Inr or Initiator start site of Transcription: Py2CAPy5, with posn +1 at the A
2. The TATA box, ~25 bp upstream of +1 ... for TBP binding
In addition, the efficiency and specificity of promoter recognition depends on short sequences usually upstream 20-200 bp of Inr ...
These short sequences are recognized by Protein Activators: Upstream Factors which usually enhance and activate transcription efficiency and (sometimes) specificity ... [Brown, Fig 8.17]
Mutagenesis and computer analysis identifies two regions showing promoter down mutations:
1. TATA box centered at -30 ... mainly a positioning element for transcription initiation, little effect on promoter strength (2-fold) or specificity
2. GC box centered at -90 ... sequence GGGCGG ... multiple copies per promoter ... binds the factor SP1 (Stimulatory Protein 1)
Both are common RNApolII promoter elements
... These and other examples are as follows:
... [Brown, Table 8.4]
Element ConSeq Factor TATA box TATAAAA TBP (Tata Binding Protein) CAAT box GGCCAATCT CTF/NF1 (CaaT binding Factor / Necrosis Factor 1) GC box GGGCGG SP1 (Stimulatory Protein 1) Octamer ATTTGCAT Oct-1/Oct-2 (Oct-2 specific to Lymphoid cells) ATF GTGACGT ATF
2. Factors that recognize the Upstream Element Sites: ... [Brown, Table 8.4]
The Upstream Factors are more or less ubiquitous and hence available to any promoter with a recognition sequence in the promoter
Sequence element positions largely unimportant; can be interchanged between different promoters ... distance between elements unimportant ... ==> looping of DNA important; protein-protein interactions important ...
Upstream Elements may be recognized by more than one Factor, eg CAAT box:
1. CTF factor family, generated by alternative splicing from single gene
2. CP1, CP2, CP3 bind CAAT boxes in different genes ... gene specificity
3. C/EBP binds GCAAT, ACF binds CCAAT ... sequence specificity
Regulation possibilities exist here as well ... Example: Histone H2B
In sea urchin testis, CAAT is bound normally and H2B is expressed
In sea urchin embryos, CAAT is bound by CDP (CAAT Displacement Protein) which prevents binding of normal CAAT binding factors -> H2B is not expressed
Thus, CDP acts like a prokaryotic operon repressor !
Octamer sequence is another example of an Upstream Element that can be bound by more than one Upstream Factor:
1. Oct-1 factor is ubiquitous and the only Oct factor in non-lymphoid cells
2. Oct-2 factor in lymphoid cells binds to Octomer to activate IG kappa light gene; this gene is not activated by Oct-1 factor in non-lymphoid cells
Thus promoter context is important ... totality of elements and factors is important
B. Sequence Elements NOT in Promoter itself:
Enhancers
... [Brown, Fig 8.17 C]
Located at variable distance from Upstream Elements considered to be in Promoter
Operational Definition: Promoter includes those Upstream Elements found in relatively fixed locations relative to Inr
Enhancers are elements whose position relative to Inr is not fixed and which function in either orientation. Usually enhancers activate ANY promoter in their vicinity.
Enhancers are also identified with genes selectively expressed in particular tissue ... thus, enhancers are responsible for much selective gene expression in eukaryotes
Enhancers in yeast are called Upstream Activator Sequences (UASs)
Example: Enhancer in virus SV40 which is composed of two copies of 72 bp sequence
This enhancer can be placed ~10,000 bp upstream or downstream of promoter for early gene expression and can be in either orientation ... and transcription occurs normally !!
But if enhancer is deleted or placed on a second DNA molecule, no transcription occurs
Enhancers most likely function by bringing Transcription Factors together into the Promoter region, via looping of the DNA
C. Regulation of Tissue-Specific Gene Transcription
... [Brown, Table 8.4]
Cell type phenotypes are largely determined
by differential gene expression:
1. some types of cells express some genes, others express other
genes
2. expression of different sets of genes leads to different paths
of cell differentiation
At which stage in gene expression is such control exerted?
Mainly at stage of initiation of transcription by RNA polymerase II and via alternative splicing events during mRNA maturation ... no evidence for Anti-Termination
This is usually done via a few, key Transcription Factors that provide common control of many target genes.
The question of regulation then becomes two
specific questions:
How do internal and external signals regulate activity of the
Transcription Factor?
How does the Transcription Factor identify the correct genes to
activate?
1. Promoter elements identify Genes to Activate:
Response Elements
... [Brown, Table 8.4]
Examples of Response Elements: ... see J. Art. 5 by Karin et al
HSE - Heat Shock
Element; GRE - Glucocorticoid Response Element
SRE - Serum Response Element; MRE - Metal Response
Element
Properties are similar to Upstream Elements and Enhancers:
Short Consensus Sequences; found usually in Promoters but sometimes in Enhancers; one Response Element usually sufficient for gene activation
Function: Response Element is a binding site for a specific protein.
Active protein acts as a Transcription Factor which activates transcription of genes with the Response Element
Example: Transthyretin (TTR) gene - encodes serum protein that binds thyroid hormone ... made mainly in hepatocytes (liver cells) ...
10 Response Elements plus TATA box ... six proximal, four in distal Enhancer
Response Elements bind tissue specific Transcription Factors:
C/EBP - classic leucine zipper ... [Brown, Fig 7.24]
HNF1 - Hepatocyte Nuclear Factor 1; homeobox protein ... [Brown, Fig 7.21]
HNF3 - "winged helix" binding domain ...
HNF4 - C2H2 zinc finger ... [Brown, Fig 7.20]
AP1 - leucine zipper binding proteins (JunB is a component of AP1) ... [Brown, Fig 7.24]
These Transcription Factors, except for JunB, are made in liver but not in kidney ...
These all assemble cooperatively, forming a multi-protein-DNA complex as the RNApolII transcription initiation complex ...
2. Transcription Factor Domains
Transcription factors must perform at least
the following two functions:
1. bind to target Enhancer and Upstream Element sequences
2. interact with other members of the Transcription Apparatus
These capabilities usually reside in separate domains in the Transcription Factor protein
Function of Enhancers and other DNA Elements: provide high concentration of Transcription Factors in vicinity of Promoter and Basal Transcription Apparatus
Bound Upstream Transcription Factors activate transcription most likely via protein-protein interactions with Basal Transcription Apparatus proteins, eg TFIID, TFIIB
Example: Yeast Activator GAL4
Regulates genes involved in galactose catabolism in yeast ...
GAL4 binds a yeast DNA Enhancer: UASG (Upstream Activating Sequence)
GAL4 has three functions: binds UASG; activates transcription; binds GAL80, another regulatory protein
GAL4 has separate domains for each of these functions
DNA binding and dimerization domains are at N-terminal end, whereas GAL80 binding and one of two transcription activation domains are at C-terminal end
Either of the two transcription activation domains has full activity by itself
Thus: separation of functions, consistent with flexible connector region joining these ...
Heterodimer proteins can be formed between different subunits (monomers) of proteins of the same class, extending the ability of these proteins to bind to different sites ...
GAL80 function:
in absence of Galactose, GAL80 binds GAL4, inactivating GAL4
GAL80 is thus a Repressor ...[Brown, Table 8.4 D]
In presence of Galactose, GAL80 is released and GAL4 can activate
transcription
Common regulatory feature: A second protein binding a Transcription Factor to regulate its activity, often via negative-acting Repressor-like activity.
3. DNA Binding Specificity and Domain Separation
of Function:
... [Brown, Fig 11.5]
The DNA-binding domain of GAL4 was substituted with the DNA-binding domain of LexA (SOS box) via a recombinant DNA chimeric plasmid experiment
Ability of this chimeric GAL4 to activate a target promoter was examined, with either the UASG element or the LexA operator (SOS box of the SOS response) placed near the promoter:
Result: The chimeric LexA-GAL4 activated transcription only from the promoter with the LexA operator attached !!
Clear demonstration of Domain separation of Function AND Specificity of DNA Binding
Many such "mixing" experiments have now been done ...
4. Yeast Two-Hybrid System: Method for determining Protein Interactions
Take advantage of the separation of DNA Binding protein domain from the Activation Function protein domain in the Transcription Factor ...
The GAL4 Transcription Factor has been used for this ...
Two Hybrid System
used to show Ras-Raf interaction, as shown below:
Ras gene as "bait" with GAL4 DNA binding domain: fusion
or Hybrid Proteins
cDNAs as "fish" with GAL4 activation domain
D. Transcription Control by Small Molecules: Lipid-Soluble Hormones
Hormones: extracellular signals secreted from one cell and travel to effect function of cells at a different locations.
Lipid-soluble Hormones: can diffuse through membranes and enter cells ... [Brown, Fig 11.4]
Examples: Cortisol, Retinoic acid, Thyroxine, Estradiol
Steroid Hormone Receptors:
When bound to Hormone, receptors are
active as protein Transcription Factors
Example: Mouse
Mammary Tumour Virus gene
Glucocorticoid Receptor activates this gene by binding four regions:
Glucocorticoid Response Element (GRE) response element
Response Elements for diffferent Steroid Hormone
Receptors have direct and inverted repeats
... [Brown, Fig 11.4]
Examples of Steroid Hormone Receptors and their Response Elements:
Response Element Receptor Glucocorticoid Response Element (GRE) Glucocorticoid Receptor Estrogen Response Element (ERE) Estrogen Receptor Vitamin D Response Element (VDRE) Vitamin D3 Receptor Retinoic Acid Response Element (RARE) Retinoic Acid Receptor
These Receptors have distinct protein regions
for DNA binding and for Hormone binding
... [Brown, Fig 11.5]
The DNA binding domain of Glucocorticoid Receptor is a C4 zinc finger motif; the Receptor is normally a homodimer ...
Hormone binding required for Receptor translocation to nucleus ...
General model: Gene Activation by Lipid-Soluble Hormones ...
E. Transcription Control by Small Molecules: Lipid-Insoluble Hormones
Lipid-insoluble Hormones can not pass through the cell membrane, yet still result in activation of specific genes via activating Transcription Factors, much as in the above.
How to do this?
Signal Transduction and Cell Surface Receptors
Basic approach:
1) Small molecule Hormone interacts with a transmembrane Cell Surface Receptor.
2) This interaction activates the Cell Surface Receptor, which in turn activates another intracellular protein.
3) This second protein can activate a third
... and so on ... eventually resulting in a Transcription Factor
binding to an Enhancer or Proximal Regulatory region, causing
activation of one or more genes ... This whole process is called
Signal Transduction: ... [Brown, Fig 11.7]
the "signal" from the Hormone at the cell surface
is "transduced" or "conducted" or "moved"
to the nucleus resulting in Gene Activation.
4) The initial hormone "signal" is
amplified during signal transduction, resulting in a cascade
of many copies of the molecules in the signal transduction pathway.
Small molecules can be part of this Signal Transduction, serving
a role as a Second Messenger, carrying the "message"
of the Hormone to the nucleus for gene activation.
Examples of Second Messengers: cAMP and Ca++
Examples of Signal
Transduction Cascade Mechanisms: ... [Brown, Table 11.2]
1) G-protein coupled receptors
2) Tyrosine kinases
3) Ser-Thr kinases
4) Ion Channels
F. Addenda: Clarification of Nomenclature
The following is a classification scheme for initiation of transcription in eukaryotic cells that in general works, realizing that there are exceptions (nearly always true in biology, which is what makes it so difficult to make biology a predictive science):
1. Sites on DNA classified by POSITION relative to genes and transcription start sites (usually Inr sites):
a. Promoter: region immediately upstream of transcription start site where HoloRNApol is assembled and initiation occurs.
Generally is the region -1 to -200 relative
to the start site.
Contains:
Inr
TATA box
Upstream Elements
Proximal Binding Sites (another name for Upstream Elements)
Note the exception for RNApolIII where BoxA,B,C are Downstream within the gene rather than Upstream.
b. Enhancers: regions upstream or downstream, near or far, in either orientation, all relative to the transcription start site, that contain several copies and/or types of binding sites for Transcription Activators.
2. Sites on DNA classified by FUNCTION in eukaryotic gene transcription:
a. Binding sites for assembly of RNA polymerase Holo enzymes.
We have defined RNA polymerase Holo enzymes to be:
Core RNA pol + TF factors = HoloRNApol
where Core RNA pol is the complex of Basal factors shown in Lodish Table 11-4 and Figure 11-28.
These Binding Sites for the Function of assembly are mainly Upstream Elements (or Proximal Binding Sites), but also include Box A,B,C
b. Response Elements: binding sites for Transcription Activators involved in tissue specificity, cell growth and proliferation, cell differentiation ... most of such "responses" result from events imposed on the cell: heat shock, presence of metals, presence of hormones, etc; in this sense, use of Response Element sites is "inducible".
Positions of Response Elements on DNA:
a. in Promoters ... they then are Upstream Elements
b. in Enhancers ... most binding sites in Enhancers are
Response Elements
Lodish, Figure 11-56 shows the TTR gene in mouse, where Response Elements are present both in the Promoter as Upstream Elements (6 such) and in an Enhancer (4 such).
Transcription Activators bind to Response Elements.
These Activators are sometimes called "factors" or "receptors" etc, e.g. Glucocorticoid Receptor and Hepatocyte Nuclear Factor.
3. DNA Sites vs Proteins that bind to the Sites:
Be clear on the distinction between SITES on the DNA and the Proteins that bind to these sites:
Sites - called "elements", "sequences" (e.g. UAS - Upstream Activator Sequences, in yeast), "boxes", "promoter", "enhancer"
Proteins - called
"factors", "receptors", "binding protein",
"activators", "inhibitors" (in eukaryotic
transcription, inhibitor proteins usually work by binding to an
activator protein, thereby inhibiting the activator, rather than
binding to DNA as in the case of prokaryotic operon control of
transcription).
| BIMM100 | Syllabus
| Sections / Off Hrs | Grading
Policy | DNASYSTEM
|
| Lectures | Journal
Articles | Study Qs | Lab
Techniques | Exams |
If you have problems or comments, send email to Doug
Smith