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3. Eukaryotic Transcription II chapter 7. transcription initiation of some eukaryotic genes is mediated by a so-called initiator sequence, which is however not very well defined - PowerPoint PPT Presentation
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McGillMcGill BIOL200 - Fall 2010BIOL200 - Fall 2010
© R. Roy, 2010© R. Roy, 2010
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McGillMcGill BIOL200 - Fall 2010BIOL200 - Fall 2010
© R. Roy, 2010© R. Roy, 2010
McGillMcGill BIOL200 - Fall 2010BIOL200 - Fall 2010
© R. Roy, 2010© R. Roy, 2010
Eukaryotic Transcription IIEukaryotic Transcription II
chapter 7chapter 7
McGillMcGill BIOL200 - Fall 2010BIOL200 - Fall 2010
© R. Roy, 2010© R. Roy, 2010
TATA sequence is very common in the TATA sequence is very common in the promoters of protein coding genespromoters of protein coding genes
transcription initiation of some eukaryotic genes is mediated by a so-called initiator sequence, which is however not very well defined
transcription of the overwhelming majority of eukaryotic genes is mediated by a highly conserved region termed theTATA box, which serves to position and orient the RNA polymerase
McGillMcGill BIOL200 - Fall 2010BIOL200 - Fall 2010
© R. Roy, 2010© R. Roy, 2010
TATA-box Binding Protein (TBP)TATA-box Binding Protein (TBP)
The conserved C-terminal domain of TBP binds to the minor groove of the TATA box DNA, disrupting the normal helix structure and thereby bending the DNA dramatically.
Curiously, although some promoters are “TATA-less”, they still require TBP for transcriptional initiation
McGillMcGill BIOL200 - Fall 2010BIOL200 - Fall 2010
© R. Roy, 2010© R. Roy, 2010
TBP is a component in all classes of TBP is a component in all classes of eukaryotic transcriptioneukaryotic transcription
McGillMcGill BIOL200 - Fall 2010BIOL200 - Fall 2010
© R. Roy, 2010© R. Roy, 2010
RNA polymerase II: it's complex!RNA polymerase II: it's complex!
formation of a pre-initiation complex is the rate limiting step of transcription
McGillMcGill BIOL200 - Fall 2010BIOL200 - Fall 2010
© R. Roy, 2010© R. Roy, 2010
Transcription factors recognize specific Transcription factors recognize specific DNA sequence motifsDNA sequence motifs
Commonly, transcription factors possess an alpha-helical domain, the so-called RECOGNITION HELIX.
The recognition helix can recognize a particular DNA sequence motif through non-covalent interactions with atoms in the bases.
Recognition usually occurs through interaction of the helix with the major groove of DNA.
bacteriophage repressor 434 homodimer
recognition helices
McGillMcGill BIOL200 - Fall 2010BIOL200 - Fall 2010
© R. Roy, 2010© R. Roy, 2010
Transcription factor binding sites can be Transcription factor binding sites can be found through linker scanningfound through linker scanning mutationsmutations
subregions of the promoter required for activation of transcription
Reporter genes facilitate the relative quantification of transcriptional efficacy
Some common “Reporters”:
-Green Fluorescent Protein (GFP)
--galactosidase (lacZ)
-thymidine kinase (tk)
-luciferase (luc)
-chloramphenicol acetyltransferase (CAT)
McGillMcGill BIOL200 - Fall 2010BIOL200 - Fall 2010
© R. Roy, 2010© R. Roy, 2010
Electrophoretic mobility shift assays Electrophoretic mobility shift assays (EMSA) indicate general DNA binding (EMSA) indicate general DNA binding
activityactivity• EMSA or gel/band/mobility shift assays
are commonly used during purification of transcription factors to assay for DNA binding activity
• A radiolabelled dsDNA segment that corresponds to a transcriptional regulatory region is used as a probe
• If a polypeptide or protein complex present in a mixture can bind to the labelled DNA the probe’s mobility in the non-denaturing polyacrylamide gel will be retarded compared to free probe
• EMSA cannot reveal the precise sequence that is bound by the protein!!!
McGillMcGill BIOL200 - Fall 2010BIOL200 - Fall 2010
© R. Roy, 2010© R. Roy, 2010
DNA binding activity can be visualized DNA binding activity can be visualized in vitroin vitro
Potential transcription factor binding region identified
through linker scanning or gel shift assays
column fractions of a cellular extract (which separates proteins based on various biochemical characteristics) are incubated with a radioactively labeled DNA fragment containing the transcription factor binding site
the samples are treated with randomly cutting DNAse I enzyme (partial digestion so roughly only one cut per labelled molecule) and the resulting DNA fragments are separated by gel electrophoresis
region protected from
DNAse I digestion by transcription
factor binding
McGillMcGill BIOL200 - Fall 2010BIOL200 - Fall 2010
© R. Roy, 2010© R. Roy, 2010
Transcription factors have a modular Transcription factors have a modular structurestructure
most transcription factors have several domains that each perform distinct functions
an example:
the GAL4 transcription factor from yeast
-> contains a DNA binding domain to bind UASGAL
-> contains an activation domain to stimulate transcription
GAL4
McGillMcGill BIOL200 - Fall 2010BIOL200 - Fall 2010
© R. Roy, 2010© R. Roy, 2010
Modular structure is very common in Modular structure is very common in eukaryotic transcription factorseukaryotic transcription factors
transcription factors can contain domains for:
DNA binding transcription activation transcription repression chromatin remodelling nuclear import protein interaction
McGillMcGill BIOL200 - Fall 2010BIOL200 - Fall 2010
© R. Roy, 2010© R. Roy, 2010
The The Drosophila Drosophila Antennapedia mutantAntennapedia mutant
wild type mutant
homeotic transformation:
for instance, mutation of the Drosophila gene Antennapedia leads to the formation of legs instead of antennae on a Drosophila head segment
McGillMcGill BIOL200 - Fall 2010BIOL200 - Fall 2010
© R. Roy, 2010© R. Roy, 2010
Homeodomain proteinsHomeodomain proteins The homeodomain was named due to its presence in several
transcription factors that give rise to homeotic transformations when mutated at particular residues
homeodomain binding sites
McGillMcGill BIOL200 - Fall 2010BIOL200 - Fall 2010
© R. Roy, 2010© R. Roy, 2010
Different types of zinc finger DNA Different types of zinc finger DNA binding domains existbinding domains exist
C2H2 types usually contain three or more finger units and bind to DNA as monomers
C4 types usually contain only two finger units and bind to DNA as homo- or heterodimersie…steroid hormone receptors
the C6 Zinc Finger transcription factor is a variation wherein six cysteine metal ligands coordinately bind two Zn2+ ions
McGillMcGill BIOL200 - Fall 2010BIOL200 - Fall 2010
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Leucine zipper proteinsLeucine zipper proteins leucine zipper proteins bind DNA exclusively as homo- or heterodimers with their extended alpha-helices, which bind the DNA’s major groove
they contain a leucine or a different hydrophobic amino acid in every seventh position in the C-terminal region of the DNA binding domain
these hydrophobic residues form a coiled coil domain, which is required for dimerization
McGillMcGill BIOL200 - Fall 2010BIOL200 - Fall 2010
© R. Roy, 2010© R. Roy, 2010
Helix-Loop-Helix proteinsHelix-Loop-Helix proteins helix-loop-helix (HLH) proteins are very similar to leucine zipper proteins, however instead of an extended alpha-helix they are characterized by two alpha-helices, which are connected by a short loop
HLH proteins contain hydrophobic amino acids spaced at intervals characteristic of an amphipathic alpha-helix in the C-terminal region of the DNA binding domain
McGillMcGill BIOL200 - Fall 2010BIOL200 - Fall 2010
© R. Roy, 2010© R. Roy, 2010
Transcription factors of unrelated classes Transcription factors of unrelated classes can bind COOPERATIVELYcan bind COOPERATIVELY
protein-protein interactionfavours formation of, and stabilises
the ternary complex (DNA Binding)
SOME TRANSCRIPTION FACTORS ACTUALLY REQUIRE COACTIVATORS…
McGillMcGill BIOL200 - Fall 2010BIOL200 - Fall 2010
© R. Roy, 2010© R. Roy, 2010
Combinatorial possibilities greatly extend Combinatorial possibilities greatly extend the potential for diversified gene regulationthe potential for diversified gene regulation
The combination of transcription factor binding sites in promoters, paired with the potential for protein-protein interactions among the transcription factors to enhance the diversity of transcriptional responses
Homo- and heterodimer formation is common among transcription factors,
ie…three transcription factors that can homodimerize or heterodimerize=> 6 different possible combinations
McGillMcGill BIOL200 - Fall 2010BIOL200 - Fall 2010
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An example: specification of floral An example: specification of floral organs in organs in ArabidopsisArabidopsis
Arabidopsis flowers consist of concentric whorls of organs: four sepals in the outer whorl surround four petals in the next whorl, followed by six stamens and two carpels.
These organs are specified by the combinatorial action of a limited number of related transcription factors of the so called MADS box class.
McGillMcGill BIOL200 - Fall 2010BIOL200 - Fall 2010
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MADS box factors specifying flower organs MADS box factors specifying flower organs are expressed in specific domainsare expressed in specific domains
MADS box transcription factors involved in floral organ formation are expressed in distinct patterns.
The expression patterns lead to the formation of distinct multimeric MADs box factor complexes in the different whorls.
The different MADS box complexes have different affinities for MADS box target DNA sequences, thus establishing differential expression of target genes.
This is best describedby an “ABC” model to explain floral organ patterning!
McGillMcGill BIOL200 - Fall 2010BIOL200 - Fall 2010
© R. Roy, 2010© R. Roy, 2010
sepal petal stamencarpel
PI
AP1 AG
SEP3
AP1AP1 PI AP3 PI AP3 AG SEP3
AP1SEP3 AG SEP
3
organ domain:
MADS box factorexpression:
MADS box factorcomplex:
AP3
The MADS box factors in floral identity:“A” function: APETALA1“B” function: APETALA3
PISTILLATA“C” function: AGAMOUS
An important accessory factor:SEPALLATA 3
note: only AP1 and SEP3 contain transcription activation domains!!!
