Most gene regulation takes place when?
transcription
most important different because prokaryotic and eukaryotic DNA?
EUkaryotes have nucleosomes that form chromatin
-many nucleosomes are positioned to block the promoter
-chromatin structure usually has to be changed inorder to allow transcription - depends on the binding of regulatory proteins
Ground state of Euk and PRok
-Prokaryotes genes are generally "on"
-Euk genes are generally "off
General transcriptional differences between prok and euk
1. In bacteria, all genes are transcribed by the same RNA polymerases. There are three involved with euks. RNA polymerase II does mRNA
2. RNA transcripts are extensively processed in euks and introns spliced
3. RNA pol II is much larger and more complex t
eukaryotic gene regulation must be able to
1. ensure that the expression of most genes is in the genome is off at any one time while activating a subset of genes
2. generate thousands of patterns of gene expression
Divide regulatory proteins into two sets:
1. large RNA polymerase II complex and the general transcription factors. to initiate transcription, these proteins interact with DNA sequences called promoter proximal elements near the promoter
2. transcription factors that bind cis acting regulatory se
promoter proximal elements
DNA sequences that proteins must interact with to initiate transcription
enhancers
may be located a considerable distance away from the promoter. they are bound by transcription factors that control the regulation of smaller subsets of genes. will usually only act in one or a few cell types in a euk.
Much of the strategy of eukaryotic transcriptional control hinges on how specific transcription factors control the
access of general transcription factors and RNA polymerase II
If sequence elements are mutated,
transcription is generally reduced
regulatory proteins posess one or more of these functional domains
1. A domain that recognizes a DNA regulatory sequence (the protein's DNA binding site)
2. A domain that interacts with one or more proteins of the transcriptional apparatus (RNA polymerase or a protein associated with RNA polymerase)
3. A domain that inte
yeast imports sugar and converts it into
glucose that can be metabolized.
GAL1, GAL2, GAL7, GAL10
encode enzymes that catalyze steps in the biochemical pathway that converts galactose into glucose
GAL3, GAL4, GAL80
encode proteins that regulate the expression of the enzyme genes.
In yeast cells growing in media lacking galactose, the GAL genes
are silent
in the presence of galactose, GAL genes are
induced.
Key regulator of GAL gene is
GAL 4 protein. best studied transcriptional regulatory protein in eukaryotes.
GAL4 regulates multiple genes through
upstream activation sequences
In the presence of galactose, the expression of GAL1, GAL2, GAL7, and GAL10 are
1000 fold or more higher than in absence
In GAL4 mutants, GAL1,2,7,10
are silent
Each of GAL1,2,7,10 have two or more GAL4 binding sites locted
upstream of its promoter
Between GAL1 and GAL10 transcription start sites, there is
118 bp region that contains four GAL4 binding sites. Each GAL4 binding site is 17 bp and is bound by one GAL 4 protein dimer.
GAL4 binding sites
necessary for gene activation in vivo. if they are deleted, the genes are silent even in the presence of galactose.
upstream activation sequences( UAS)
because GAL4 enhancers are located upstream of the genes they regulate
activation domain
a distinct domain of the GAL4 protein that is required for regulatory activity.
GAL4 has two domains
1. for DNA binding
2. activating transcription
In order to find the organization of the gal4 protein
-series of elegant experiments
-test the DNA binding and gene activation of mutant forms of the protein in which parts had been either deleted or fused.
-investigators could deterimine whether a part of the protein was necessary for a particular function
reporter gene
monitors the expression of gal genes and other transcription factors. a reporter gene's level of expression is easily measured.
-linked to the regulatory sequences that govern the expression of the gene being investigated
-the expression of the reporter g
To investigate the control of GAL gene expression,
the coding region of one of thse reporter genes and a promoter are placed downstream of a UAS element from a gal gene. Reporter gene expression then becomes a readout of gal4 activity in cells
when a form of gal4 lacking the activation domain is expressed in yeast
the binding sites of UAS are occupied but no transcription is stimulated
the same is true when other regulatory proteins lacking activation domains are expressed in cells bearing reporter genes with their binding sites
when a form of gal4 lcking the DNA binding domain is grafted to the DNA binding domain
the hybrid protein now activates transcription from lexA binding sites. "Domain swap
domain swap
show that transcriptional activation function of the gal4 protein resides in two small regions about 50 to 100 amino acids in length. these two regions form a separable activation domain that help recruit the transcriptional machinery to the promoter
Many eukaryotic transcriptional, regulatory proteins are modular proteins having separable domains for
DNA binding, activation or repression, and interaction with other proteins
Gal4 activity is physiologically
regulated
How does gal4 become active in presence of galactose?
key clues came from analyzing mutations in the gal80 and gal3 genes.
in gal80 mutations
gal structural genes are active even in the absence of galactose. suggests that the normal function of the gal80 protein is to somehow inhibit gal gene expression
gal80 binds to gal4 protein with high affinity and directly inhibits gal4 acitvity. binds to
in gal3 mutations
the gal structural genes are not active in the presence of galactose; suggesting that gal3 normally promotes expression of gal genes
the role of the gal3 is to release gal structural genes from their repression by gal80 when galactose is present
both a se
gal3 gal80 gal4
all part of a switch whose state is determined by presence or absence of galactose.
gal4 can also affect transcription in insects, humans, etc.
suggests that biochemical machinery and mechanisms of gene activation are common to a braod array of euks and that features revealed in yeast are generally present in other euks
in euks, activators work
indirectly
euk activators recruit rna polymerase through two mechanisms
1. activators can interact with subunits of the protein complexes having roles in transcirption initation
2. activators can recruit proteins that modifty chromatin structure allowing rna pol II and other proteins access to the dna
gal4 has both activities
gal4 binds TBP to a site in its activation domain. through this binding interaction, it recruits the
TFIID complex and in turn RNA polymerase II to the promoter.
mediator complex
another way that gal works to activate gene expression
it is a large multiprotein compelx that in turn directly interacts with RNA pol II to recruit it to gene promoters. the mediator complex is an example of a coactivator
coactivator
a term applied to aprotein or protein compex that facilitates gene activation by a transcription factor but that itself is neither part of the transcriptional machinery nor a dna binding protein
eukaryotic transcriptional activators often work by recruiting parts of the transcriptional machinery to
gene promoters
A second mechanism for influencing gene transcription in eukaryotes modifies the local chromatin structure around regulatory sequences
review chromatin structure and then consider how it can change and how these changes affect gene expression
prok DNA is relatively naked compared to
euk DNA. makes it very easy for RNA polymerase to attach. euk chromosomes are packaged in chromatin which is composed of DNA and proteins.
prok genes are generally accessible adn "on" unless repressed,
euk genes are inaccessible and "off" unless activated.
epigenetic inheritance
chromatin structure must be inherited. defined operationally as the inheritance of chromatin states from one cell generation to the next.
nucleosome positions can change on the DNA from cell to cell over the life cycle of an organism
transcription is repressed when the promoter and flanking sequences are wound up in the nucleosome, preventing the intiation of transcription by RNA pol II.
activation would require nudging the nucleosome away from
promoter
chromatin remodeling
the changing of nucleosome positioning. an integral part of euk gene expression.