Exam 2: Ch 7 Eukaryotic Gene Control Flashcards
2 features of transcription control common to bacteria and eukaryotes
protein binding regulatory DNA sequences (control elements) associated with genes
specific proteins bind to a gene’s regulatory sequences to determine where transcription will start and either activate or repress transcription
what control do eukaryotes have but bacteria dont
chromatin structure regulation
promoter
DNA sequence that specifies where RNA polymerase binds and initiates transcription
controlled by DNA binding proteins
transcription factor
transcriptional regulatory proteins that either activate or repress transcription depending on association with other proteins
upstream
opposite the direction of transcription
downstream
in the same direction as transcription
TFs that regulate expression of protein binding genes can bind to control elements found…
very far away from the promoter they regulate
transcription of a single gene can be regulated by the binding of multiple different…
TFs to alternative control elements
reporter gene
analyze gene control regions by prepping recombinant DNA with a fragment to be tested containing a reporter gene
ex. luciferase (light generating) or GFP
enhancer
distant transcription control region
3 different …. catalyze formation of different RNAs
RNA polymerases I, II, III
RNA polymerase I
located in nucleolus
transcribed genes encoding precursor rRNA (pre-rRNA)
RNA polymerase II
transcribes all protein-coding genes
RNA polymerase III
transcribes genes encoding tRNAs
5SRNA
stable RNA
the largest subunit in RNA pol II has an essential carboxyl terminal repeat
carboxyl terminal domain (CTD), must have at least 10 copies in yeast to survive
RNA Pol II initiates transcription at DNA sequences corresponding to..
the 5’ cap of mRNA
3 DNA sequences can function as promoters for RNA Pol II
TATA boxes
initiators
CpG islands
TATA boxes
a conserved sequence upstream from the transcription site
required for efficient transcription in viral genes and cellular protein coding genes in cell cycle
initiator sequences
alternative promoter element with a cytosine at -1 and adenine at transcription start site (+1)
CpG island
relatively rare promoter sequence in mammals that contain high numbers of CG sequences
transcription initiated in both directions even though mRNA only made from sense strand (divergent transcription)
why are CpG islands rare in mammals
spontaneous deamination of C generates thymidine CG –> TG
initiation by RNA pol II requires…
several initiation factors called general transcription factors
position Pol II at transcription start sites and help separate DNA strands so template can enter active site
examples of general transcription factors
TFIIA TFIIB
contains TATA binding protein (TBP)
multimeric proteins
preinitiation complex
complex of Pol II and general transcription factors bound to a promoter and ready to initiate transcription
how does the preinitiation complex form with a TATA box
TBP binds to the TATA promoter and bends the DNA
TFIIB binds
Pol II-TFIIF binds
2 more general TF bind TFIIE and TFIIH
TFIIH exhibits _____ activity
helicase
uses energy from ATP to unwind DNA at start site to allow Pol II to begin transcription
also, DNA repair activity
in vivo transcription initiation by RNA Pol II requires…
TFIIA as well
_______ factors regulate the initial stages of transcription in the promoter-proximal region
elongation
in metazoans, at most promoters Pol II pauses after transcribing 20-50nt due to..
binding of negative elongation factor
DSIF binds and elongation continues
viral protein Tat allows
Pol II to read through transcription blocks
used in HIV
what are linker scanning mutations used for
pinpoint sequences in a regulatory region that control transcription
promoter-proximal elements were discovered this way
can enhancers be upstream or downstream from a promoter, within an intron, or downstream from the final exon of a gene?
yes
many enhancers are cell-type _____
specific
enhancer controlling Pax6 in retina is in intron between exons 4 & 5
enhancer controlling Pax6 in pancreas is located upstream of exon 0
do enhancers and promoters work when inverted?
yes
two techniques for detecting cognate proteins that bind to a regulatory element
DNase I footprinting
electrophoretic mobility shift assay
activators are composed of distinct ______ _______
functional domains
DNA binding domain
activation domain
DNA binding domain
binds to specific DNA sequences
activation domain
interacts with other proteins to simulate transcription from a nearby promoter
constitutive expression
unregulated, abnormally high expression
results from inactivation of a repressor
2 domains of repressors
DNA binding domain
repression domain
recognition helix
alpha helix that binds major groove of DNA
homeodomain
TF conserved DNA binding motif
zinc finger
TF structural motif with regions that fold around zinc
C2H2 zinc finger
C4 zinc finger (nuclear receptor)
leucine zipper proteins
TF binding domain made of leucine dimers
grip DNA like scissors
a diverse group of _____ _____ sequences can function as activation domains
amino acid
many TFs contain activation domains marked by an unusually high percentage of particular amino acids ____ ____ and most other yeast TFs ahve activation domains that are rich in _____ ____ ____
GAL4, CGN4
acidic amino acids (aspartic acid, glutamic acid)
acidic activation domains
capable of stimulating transcription in almost all types of eukaryotic cells
must be bound to a protein co-activator
example of TF with an acidic activation domain
mammalian CREB protein phosphorylated when there are high levels of cAMP
regulated phosphorylation required for binding of co-activator CBP, which results in transcription of genes whose control regions contain CREB binding site
nuclear receptors may have ____-binding domains
ligand
binding of ligand activates domains by inducing conformational change
combinatorial complexity of TFs
like a combination lock, some TFs bind in a certain order and must all be present for their effect to work
expands number of DNA sites these TFs can activate transcription and the numbed of ways they can be regulated
enhanceosome
large DNA-protein complexes assembled from TFs binding multiple binding sites in an enhancer
why is considerable leeway in spacing between regulatory elements in transcription control regions possible?
flexible regions connect DNA-binding domains and the activation/repression domains in TFs
ability of interacting proteins on distant sites to produce DNA loops
there are _ general methods repressors and activators that bind to specific sites in DNA regulate expression of associated protein-coding genes
3
1 way regulatory proteins regulate expression of genes by acting in concert
modulate chromatin structure to inhibit or stimulate ability of general TFs to bind to promoters
mediator
mediator of transcription complex
regulatory proteins interact with this structure that binds to Pol II and directly regulates assembly of transcription preinitiation complexes
after chromatin in open form
formation of heterochromatin silences gene expression in 3 regions
telomeres
centromeres
other
heterochromatin
dark staining and highly condensed areas of chromatin
less accessible DNA for TFs
central mating type locus of yeast, and 2 additional loci
MAT, actively transcribed and expresses TFs that control the mating type (alpha or a)
HML and HMR located near the right and left telomere, and are silent (nontranscribed copies of alpha/a genes)
the alpha/a genes are transferred from HML/HMR to MAT
how is transcription of HML/HMR repressed?
silencer sequences located nearby
condensed chromatin sterically blocks TFs from interacting with DNA
the telomeres of yeast chromosomes behave like…
silencer sequences
repressors can direct histone _______ at specific genes
deacetylation
histone deacetylation
unacetylated lysines pos charge interacts with phosphates on DNA, other histones, and other chromatin associated structures
causes condensation that TFs cant bind to
how do activation domains of DNA-binding activators function
by binding co-activator complexes
activators can direct histone acetylation at specific genes
what does activator directed hyperacetylation of nucleosomes near a promotor region do
opens chromatin structure to facilitate binding of other proteins required for transcription initiation
generates binding sites for bromodomains
chromatin remodeling factors help…
activate or repress transcription
push DNA into nucleosomes to facilitate binding of TFs
chromatin remodeling factors required for many processes…
transcription control, DNA replication, recombination, and DNA repair
transcriptional repression
yeast two-hybrid system
exploits flexibility in activator structures to identify genes whose products bind to a specific protein of interest
tests if 2 proteins bind to each other
use color assay to detect protein binding
how does yeast two-hybrid system work
yeast vector expresses 2 hybrid proteins
DNA binding domain + linker + bait domain (protein of interest)
second hybrid protein: activator domain + linker + fish domain (proteins that may or may not interact with bait)
only transformed cells expressing the bait hybrid interacting with the fish hybrid can activate transcription (b/c activation domain on fish)
which TFs are expressed in diff cell types and how much is determined by…
regulatory interactions btw TF genes that occur during development and differentiation of the cell type
extracellular signals
how do signals regulate TFs
interactions between extracellular domains of transmembrane receptors and ligands activate intracellular domains
transmits signal to nucleus where TFs are
2nd major group of extracellular signals
small, lipid soluble hormones that diffuse through plasma and nuclear membranes to interact w/ TFs they control
steroids, retinoids, thyroid hormones
nuclear receptor superfamily
intracellular receptor for most of the lipid hormone signal molecules
function as transcription activators when bound to ligand
all nuclear receptors have 3 common domains
N-terminal region: activation domain
zinc finger
C-terminal region: hormone binding domain (bound = activation, unbound may repress)
response element
characteristic nt sequence of DNA sites that bind nuclear receptors
small repeat sequence like 6bp for estrogen are same for other receptors like thyroid hormone…differ by nt spacers
specificity of response elements afforded by
spacing between the repeats of the sequence
heterodimeric nuclear receptor functions when hormone binds/doesnt bind
located only in nucleus
with hormone, activate transcription by directing histone hyperacetylation
without hormone bound, repress transcription by directing histone deacetylation at nearby nucleosomes
homodimeric nuclear receptor
found in cytoplasm when not bound to ligand (cant interact with response elements)
hormone binding directs transportation to nucleus, where it interacts with response elements, and activates transcription by interacting with chromatin-remodeling and histone acetylase complexes and mediator
regulation of Pol II transition from initiation to elongation
Pol II paused around 200bp of transcription start site until TF binds
allows rapid response: genes are paused until an emergency where a TF binds and high expression occurs
ex. heat shock gene
epigenetic
inherited changes in the phenotype of a cell that do not result from changes in DNA sequence
post translational modifications of histones and DNA
epigenetics and CpG islands
active CpG island promoters have unmethylated C’s
in differentiated cells, a few CpG island promoters are methylated, which triggers chromatin condensation
epigenetics and histone modification
acetylation/deacetylation is turned over quickly b/c held in equilibrium by acetylase enzymes
methylation turned over slowly and is good for propagating epigentic information
are methylated histones passed down?
yes, to approximately half of daughter cells
maintains methylation of these histones in certain areas of chromosome
polycomb proteins
epigenetic mark essential for repression of genes in specific cell types and all the subsequent cells that develop throughout life
repress Hox genes
opposite are Trithorax proteins (express Hox genes)
Hox genes
direct development of tissues and organs in the embryo
important in cell growth and division (often mutated in cancer)
polycomb proteins repress these genes early in embryogenesis
trithorax proteins express these genes
only Hox genes in cells where they were initially repressed by polycomb proteins…
remain repressed throughout life
same Hox genes in other cells remain active in the presence of same polycomb proteins
polycomb subunits
PRC1: ubiquitin ligase that prevents Pol II elongation
PRC2: histone deacetylase and histone methyl transferase
noncoding ___s can direct epigenetic repression in metazoans
RNAs
protein bound
ex. X-chromosome inactivation in females by Xist RNA-protein complex
regulation of flowering time
FWA gene encodes a homeodomain TF that regulates flowering time in response to temp so plants don’t flower until the warm spring
repressed by CHH methylation
RNA Pol I
uses a core element and upstream control element for transcription
Pol III
internal promoter elements A box and B box
C box promoter
p53 represses Pol transcription
