Lecture 18 Flashcards
ability to change sex is called
sequential hermaphroditism
In eukaryotes DNA is packaged in nucleosomes into
chromatin
inaccessible to proteins; ground state is “off”
Prokaryotes → circular genome not bound to histone proteins
In eukaryotes there are ____ chromosomes, cells, cell types
Multiple
→ timing and pattern of expression varies among them
In eukaryotes there are ___ RNA polymerases
3 different
In eukaryotes Genes are split (____ interrupted by _____)
exons interrupted by introns
no introns in prokaryotes
In eukaryotes pre-mRNA is __, __, and ___ from nucleus
(alternatively) spliced, processed (cap and tail), and transported from nucleus
No coupled transcription/translation line in pro (Pro → no nucleus)
In eukaryotes there are many genes, ____ gene density, ___ distances between genes
low gene density (actuall coding seq low % of DNA)
long distances between
Operons are absent or very rare (Pro → operons)
ways to regulate gene expression in eukaryotes
1) transcriptional regulation
2) mRNA processing
3) regulation of mature mRNA
4) translation
5) post- translation
How is the complexity of cell type specific expression achieved?
- DNA regulatory elements, and protein regulatory factors in eukaryotesa. Alternative promoters
b. Enhancers
c. Insulators
DNA regulatory elements are ___ acting
these include
cis
core promoters
Enhancers, silencers (proximal and distal)
insulators
core promoters are
binding sites for RNA pol and general transcription factors (GTFs) → creates basal transcription apparatus
Enhancers, silencers (proximal and distal) are
binding sites for transcription factors
that through bending of DNA interact with basal transcription apparatus
insulators are
binding sites that impose barriers or topological domains for enhancer activity
Protein regulatory factors are ___ -acting
these include
trans
transcription factors
coactivators / corepressors
transcription factors
(both activators and repressors) directly bind enhancers
coactivators / corepressors interact with
transcription factors but do not bind DNA directly
In eukaryotes is there one promoter-one gene
NO
single gene can have many promotes (>1)
___ of human genes have 2 or more alternative promoters
> 50%
Alternative promoters produce different ____ and ____
pre-mRNAs
different protein isoforms
protein isoforms
functionally similar proteins with similar but not identical amino acid sequence
transcription starts at different points
Duchenne muscular dystrophy (DMD)
genetic muscle disorders caused by
loss of function of dystrophin protein in skeletal muscle
(DMD) gene spans ___ , up to ___ exons, and encodes up to ___ amino acids
2.2 Mb
79 exons
3,678 amino acids
DMD isoforms from promoters
7 isoforms from 7 promoters
7 different promoters are active in different cell types and tissues
7 resulting protein “isoforms” vary from full-length (427 kDa) to only 71 kDa
RNA pol II must bind a core promoter and GTF…but promoter choice influenced by ___
specific transcription factors that bind promoter-proximal (brown boxes in figure) and distal enhancers (green boxes in figure)
[Promoter use depends on ‘enhancers’ and cell type-specific regulatory proteins]
Loss of Pax6 causes ____
Because ___
eye defects (conserved across species)
Pax6 encodes a transcription factor
“master controller” of eye (and CNS) development
BUT not all Pax6 mutations are in the coding sequence
Some patients with eye defects imacted by PAX6, have no mutation in PAX6 gene, but lack ____
thus, defect is due to ____
enhancer due to chromosomal breakage far downstream (~125kb)
due to mis-expression of gene, because of enhancer loss
Enhancer of Shh is ___
____ in Shh causes defects
ZRS
Single base substitutions in Shh enhancer cause defects
Shh: sonic hedgehog gene, regulates
vertebrate
digit identity and number
ZRS: Zone of polarizing activity Regulatory Sequence, 800 kb upstream!
highly ____
conserved enhancer controlling Shh activity during development
Representative human phenotypes caused by base substitutions in ZRS
hand changes
limb defects
If enhancers can stimulate any gene, even in distant areas, what keeps activity in check?
Insulators
Insulator is a DNA sequence that
blocks enhancer if bound by insulator-binding protein (CTCF in vertebrates)
CTCF-bound insulators form
chromatin loops called → Called Topologically Associated Domains (TADs)
“neighborhoods” of regulatory elements & genes
If insulator is not in between inhansor and promoter
insulator binds to proteins CTCF
TAD can form - allows enhancer and promoter to interact
If insulator is between enhancer and promoter
“insulate effects”
long-range transcriptional activation in eukaryotes
(with activators, enhancers, transcriptional machinery, repressors, insulators)
Transcriptional activators bind enhancers
Activators recruit transcriptional machinery & bend DNA long distances
Binding by repressors silence enhancers
Binding of insulators blocks enhancers between neighborhoods
The yeast GAL system is a ____
that _____
model of how eukaryotes coordinate gene expression (no operons!)
includes, but goes beyond, mere cis-acting sites and trans-acting factors (chromatin structure!)
GAL4 system contains many ____ motifs
zinc fingers - DNA binding motifs
In the yeast GAL system
genes include
4 genes encoding enzymes that metabolize galactose for energy
3 regulatory genes located at a distance
GAL4, GAL3, GAL80
All genes have individual promoters, coordinately controlled by → UAS enhancers
(Upstream Activator Sequence (UAS))
similarities and differences between Yeast GAL system vs. E. coli lac operon
similarities:
- protein-DNA interactions (think: what’s true of E.coli and elephants, sensu Jacob/Monod)
- Inducible metabolic pathway regulated by substrate
differences:
- coordination arises from enhancers, not operon organization
- epigenetic modification
How GAL4 system works
GAL4 has two domains (activation (dimer) and binding)
GAL4 activator binds UAS
gal80 (continuously expressed) binds to and blocks activation domain
in presence of galactose, GAL3 releases GAL80
GAL4 activation domain controls
expression of structural genes
GAL80 mutants that cannot bind GAL4
constitutively express structural genes
GAL3 mutants that cannot bind GAL80 are
uninducible
GAL4 exemplifies an activator protein binding an enhancer to recruit transcriptional machineryover long distances by
bending DNA
Glucose overrides induction by
galactose
In presence of glucose, the Gal system is repressed despite the presence of GAL4, due to
an epigenetic modification of histones (deacetylation by Tup1-Mig1 complex - binds to site present) (disassembling histones)
this works (in general) is through alternative splicing
Estimated that ____ are alternatively spliced
95% of human genes with multiple exons
DNase I hypersensitive sites
regions around the genes become highly sensitive to the action of DNase I durring transcrption
chromatin-remodeling complexes
(ex. SWI-SNF)
alter chromatin structure without altering the chemical structure of the histones directly
bind directly to particular sites on DNA and reposition the nucleosomes, allowing other transcription factors and RNA polymerase to bind to promoters and initiate transcription
histone code
modifications have sometimes been collectively called this
The tails of histone proteins are often modified by the addition or removal of
- phosphate groups
- methyl groups
- acetyl groups.
- or ubiquitination
mediator
interacts with RNA pol
Many enhancers are themselves transcribed into short RNA molecules called
enhancer RNAs (eRNAs)
Sometimes a number of enhancers are clustered together to form a
super-enhancer
response elements
are regulatory sequences
short stretches of DNA that typically contain the same consensus sequences at varying distances from the genes being regulated
If the same response element is present at multiple genes, it allows all those genes to be activated by the same stimulus. (ex. heat-shock elements)
gene regulation through RNA splicing example
sex determination in fruit flies.
Sex differentiation in Drosophila arises from a cascade of gene regulation
