Final Exam Flashcards
1
Q
Chromatin
A
DNA+histone
2
Q
Histone
A
Positively charged, DNA is negatively charged, interaction is favorable
DNA is wrapped around histones to form nucleosomes
3
Q
Nucleosomes
A
Most basic unit of compaction
4 histones (2xH2A, 2XH2B, 2xH3, 2xH4) + 146bp DNA
4
Q
What is the effect of genes being so compacted?
A
Many genes are not accessible by the replication machinery and the chromatin has to be unwound in order for genes to be turned on. Many genes are naturally off depending on chromatin state. A lot of energy is needed to open chromatin
5
Q
Why are different genes on in different cells?
A
Cell fates are determined and maintained by transcriptional and epigenetic mechanisms
6
Q
Prokaryotes vs eukaryotes
A
Prokaryotes - default state is on, regulated step is the repressor protein
Eukaryotes- default state is off, regulated state is turning them on at different times/space
7
Q
Six main mechanisms of eukaryotic gene regulation
A
- Transcription initiation - occurs in the nucleus, determines if, when, and how much DNA is produced
- RNA processing - different size of poly A tail
- RNA transport
- RNA stability
- Transcription efficiency
- Protein activation/modification
8
Q
Promoter
A
The main feature of many eukaryotic promoters is a TATA box. The TATA box is the binding site for TBP, one of the components of the basal transcription factor TFIID. After RNA pol II recruitment, transcription can initiate 30 bp downstream from the TATA-box
9
Q
Promoter proximal elements
A
Usually found just upstream of the TATA box, 100-300 base pairs upstream of the transcription start site
10
Q
Enhancer
A
- distance independent
- upstream or downstream
- orientation independent
- control the expression of a few genes, like cell specific genes
- control the expression of a few genes, like cell specific genes
11
Q
Locus control region (LCR)
A
Highly specialized enhancer elements that regulate the transcription of multiple genes packaged in complexes of related genes. Proteins bind to the enhancer —> enhancer turns target gene on
12
Q
Transcription factors
A
Proteins that bind the cis-DNA, help RNA Pol II carry out transcription
13
Q
Basal (or general) transcription factors
A
Necessary and sufficient for transcription at promoter and promoter proximal elements, part of the RNA pol II holoenzymes, bind to the promoter
14
Q
Regulatory transcription factors/enhancer binding proteins/activators
A
- bind enhancers away from the promoter region
- much more specific than basal transcription factors
- each regulates a much smaller subset of genes
- a given factor only functions in a few cell types
15
Q
Different class of transcription factors?
A
16
Q
Pioneer factors
A
The first to bind to regulatory modules, and their binding facilitates the binding of additional transcription factors, activators and repressors
17
Q
Coactivators and mediators
A
Don’t bind to DNA directly, but bind to proteins already bound to DNA, lack DNA binding domain
18
Q
Coactivators
A
Similar to transcription factors in that they enhance transcription. They bind transcription factors bound to enhancers to facilitate transcription.
- recruit protein complexes involved in transcription to the promoter
- recruit proteins that modify chromatin structure, allows RNA pol II and other proteins to access the DNA
19
Q
Mediator
A
A co-activator, large protein complex that links transcription factors bound to enhancers and the basal transcription apparatus bound to the promoter —> 31+ subunits, 1.3 MD
20
Q
Corepressors
A
Similar to coactivators in that they do not have a DNA binding domain. Once they bind to a DNA bound transcription factor, they have a negative affect on transcription
21
Q
How do transcription factors activate transcription?
A
- Direct interaction with the basal transcription machinery
- Slide nucleosomes and open up chromatin to make promoter accessible
22
Q
Yeast GAL4/UAS system
A
- to make use of extra cellular galactose, yeast imports the sugar and converts it into a form of glucose that can be metabolized
- 5 genes in the metabolic pathway
- 3 regulatory genes: GAL3, GAL4, and GAL80
- GAL4 is a sequence-specific DNA binding protein - the best studied transcriptional activator in eukaryotes
23
Q
GAL4
A
A sequence -specific DNA binding protein, the best studied activator protein in eukaryotes
Sequence-specific DNA binding protein that binds to the upstream activating sequences
Enhancer binding protein
2 DNA binding regions - each GAL4-binding site is 17 by long and bound by one GAL4 protein dimer.
There are two GAL4-binding sites upstream of each gene
If these sites are deleted, the genes are silenced, even in the presence of galactose —> absolutely necessary
24
Q
GAL80
A
- a repressor of GAL4
- binds to GAL4 an inhibits its activation domain
- always transcribed so it is always keeping GAL4 inactive
- GAl 3 binds to galactose and ATP, it undergoes an almost Eric change that promotes binding to GAL80
- GAL3 binding to GAL8- in turn causes GAL80 to release GAL4
- GAL3 is thus both a sensor (senses galactose) and inducer (induces GAL4 activation)
- GAL4 can interact with other transcription factors and RNA pol II to activate transcription of its target genes
25
GAL 3function
GAL4+GAL80 = no transcription
GAL80 + Gal 3 = transcription
GAL3 bind galactose and ATP, it undergoes an allosteric change that promotes binding to gal80
GAL3 is thus a sensor and an inducer
GAL4 can then interact with other transcription factors and RNA pol II to activate transcription of its target genes
26
What is the function of modular domains in regulatory transcription factors?
Concerted regulation of cellular physiology
Breakdown can occur differentially
27
Homo or heterodimers
Most transcription factors function as either homo or heterodimers, interaction is different depending on structure and sequence
28
Unique DNA binding properties (domains) of transcription factors
Helix-turn-helix motif, Zinc finger motif, leucine zipper motif, helix-loop-helix motif
29
Combinatorial action
There isn’t just one protein that binds to a transcriptional unit, need a lot of elements
30
Enhancer-blocking insulator
- regulatory elements positioned between a promoter and an enhancer
- prevents the promoter from being activated by the enhancer
- binds the insulating sequence
- has to be between the enhancer and the promoter
31
Lac operon discovery
Jacob, Lwoff, Monod. Placed more emphasis on dynamic activity and mechanisms than on structure
32
Hallmark of a good genome
Allow organisms to adapt to changes in the environment
33
Properties of genetic switches
1. Sensor - recognizes environmental conditions in which the transcription of the relevant genes is activate or repressed
2. Effectors - toggle on or off, like a switch, the transcription of each specific gene or group of genes to respond to environmental conditions
34
Promoter
The DNA segment that RNA polymerase bind and initiates transcription- determines where transcription begins
35
Repressor
Example of negative control, keeps the gene turned off
36
Activator
Example of positive control, enhancing/promoting/ turning on the gene
37
Operator
Binding site for repressor
38
Allosteric factors
Control the ability of activator or repressor proteins to bind to their DNA target sites
39
Allosteric site
Acts as a sensor that sets the DNA-binding domain in one of two modes: functional or nonfunctional, controlled by allosteric factors
40
Effect of inducer
Usually repressor is bound to the gene, blocking transcription
Allosteric inducer binds, repressor can no longer effectively interact with DNA
Transcription can occur
41
Corepressor
When it is bound to the repressor, promotes silencing
42
Allosteric inducer effect
Activator can’t bind on it’s own, when the effector is bound —> conformational change, transcription
43
Lac operon
Glucose is the preferred energy source, when lactose is present and glucose is not, genes necessary for lactose metabolism are turned on
44
Permease
Transports lactose in
45
B-galactosidase
Cleaves lactose into maltose and glucose
46
I
Lac repressor
47
P
Lac promoter
48
O
Lac operator
49
Z, Y, (A)
Genes transcribed when lactose is present
50
Lactose is present, glucose is not present
Repressor bound by allolactose, can not longer bind to the operator
51
Glucose and lactose is present
Derepresses lac operon, still need positive control
52
CAP
Catabolism activator protein, associates with cAM at low glucose concentrations and binds to the lac promote to facilitate RNA polymerase action, binds upstream of the promoter
53
cAMP
usually low cAMP levels, when ATP is broken down (low glucose) cAMP is produced
54
Catabolite repression
The inactivation of an operon/repression of transcription caused by the presence of large amounts of the metabolic end product of the operon, the repressor of the transcription of lactose -metabolizing genes in the presence of glucose is an example of catabolite repression
55
How does CAP promote transcription?
Binds upstream of the promoter, bends DNA, promotes binding of RNA polymerase
56
How does the lac repressor work?
Several binding sites, loops DNA away from where the promoter is so nothing can bind
57
IPTG
Looks similar to lactose, but its not broken down. Turns on lac operon transcription
58
X-GAL
If the lac operon is on —> blue
59
Constitutive mutation
A change in a DNA sequence that causes a gene that is repressed to be expressed continuously
60
S -silences
Always turns of transcription
61
DNA footprinting assay
A techniques to study occupancy of DNA -binding proteins to DNA
Label DNA with something fluorescent or something radioactive
1 sample - add no protein, the other - add your protein
Your protein will bind to the DNA
Add an enzyme that cleaves DNA in regular intervals
Ladder of fragments - your portion won’t be cut up
62
Chromatin ImmunoPrecipitation-seq
Looking for the presence of an accumulation of DNA to indicate that that is where your protein is binding
Isolate the DNA, use an antibody against your protein of interest to isolate it
At the end of your tube you only get the DNA that is bound to your protein
Pile up of the sequences that are bound by your protein - higher peak, more DNA
63
Chromatin ImmunoPrecipitation-PCR
Primers against your specific gene of interest, look at if proteins are bound in different conditions
64
Tryptophan operon
Gene order corresponds to reaction order in the bio synthetic pathway
65
Trp repressor
Binds DNA when tryptophan (corepressor) is present
66
Attenuation
TrpL -leader “mini gene”, translation determines transcription of everything downstream
When the mRNA is transcribed, immediately loaded on to ribosome to be translated
High abundance of trp - creates a hairpin that ends synthesis
Only 3,4 hairpin affects rna polymerase
67
How does repression and attenuation work together?
Transcription necessary for attenuation
68
Riboswitch
A segment of the mRNA binds a small regulatory molecule
Common in bacteria, regulating the expression of about 5% of genes, including genes that synthesize amino acids, nucleotides, vitamins, and other essential molecules
Bacteria riboswitches regulate transcription and translation and can alter the stability of mRNA
69
vitamin B1 bioswitch
Vitamin controls transcription, translation
MRNA made, part of leader sequence for vitamin B1
Low B1 —> mRNA folds into structure that forms an anti termination loop
B1 present - binds to riboswitch —> termination loop
70
Translation riboswitch
Prevents association of mRNA with the ribosome, prevents shine Delgado sequence to associate with mRNA
71
Heat-shock
Turn on genes for enzymes that degrade denatured proteins and chaperones that help with thermal stress
72
Sporulation
B. Subtil is forms heat and desiccation resistant spores under stressful conditions
73
Alternative sigma factors
Control of a large number of genes in bacteria is done through these
74
Signaling cascades
Sigma factors can turn each other on
75
UAS
Enhancers in the GAL4/UAS system
76
GAL4 function
Binds to mediator complex which in turn recruits RNA pol to promoters
Also binds and recruits promoters to basal transcription machinery
77
hack transcription to understand biological processes (eukaryotes)
gfp next to enhancer that is only expresses in neurons - expressed in the brain
78
chromatin structure
the compaction of chromatin has important implications for the regulation of genes and their accessibility
79
mechanisms to alter chromatin structure
- moving nucleosomes along the DNA (chromatin remodeling)
- post-translational modification in the histone tails
- replacing the common histones in a nucleosome with histone variants
80
open promoters
associated with constitutively active genes, such as housekeeping genes encoding proteins vital for basic cellular functions. Have an NDR and no TATA box
81
nucleosome-depleted region (NDR)
present in open promoters
82
covered promoters
genes whose transcription is regulated, either in an inducible, developmental, or cell-type-specific manner. Transcription of these genes is blocked until nuclosomes are displaced or removed from the promoter to allow transcription activators to bind and recruitment of RNA polymerase II binding
83
there is an anti-correlation between the position of the nucleosome and what?
the openness of the transcriptional start site and the expression of levels of the gene
84
DNAse I hypersensitive sites
used to amplify which parts of the DNA are occupied by nucleosomes or proteins. DNAse I cuts DNA only when this is naked/not protected by proteins. After cutting the DNA, the fragments are sequenced and mapped to the corresponding regions in the genome. The pileups of sequences look like peaks, and the absences look like valleys. the DNA that is naked/unoccupied is enriched (peaks)
85
micrococcal nuclease sites
used to identify which parts of the DNA are occupied by nucleosomes
mnase I cust around the nucleosomes
the DNA that was wrapped around the nucleosomes is enriched. finely cuts, destroys the DNA not around nucleosomes
86
If the nucleosome is on the transcriptional start site
the promoter is closed to transcription factors, the gene is not expressed
87
if the transcription factor is on the transcription start site,
the nucleosome is shifted
88
chromatin remodeling complexes
change the nucleosome density or position by sliding the nucleosome away from the enhancer/promoter or by displacing it all together to another piece of DNA
89
chromatin remodelling families
- SWI/SNF
- ISWI
- INO80
-ChD80
90
how are nucleosomes removed during cellular remodeling?
uses ATP, can open and close chromatin
91
why is nucleosome remodeling important?
crucial for everything from development to disease
92
the histone code
histones have a tail that stick out of the nucleosome, these amino acids can be post-translationally modified with chemical groups like acetylation and methylation, the same amino acid can have more than one modification, some only one
93
writers
place the methyl group on the tails
94
erasers
remove the methyl groups
95
readers
proteins that specifically bind the modified amino acid
96
euchromatin
genes expressed
97
heterochromatin
genes not expressed, can be turned on
98
facultative heterochromatin
genes are off but they can be turned on given the right signals
99
constitutive heterochromatin
always turned off
100
hetero chromatin can interact with the nuclear lamina
no genes that encode for proteins, anchor the DNA to the lamina
101
acetylation of lysine
acts to neutralize the positive histone charge, activates gene expression by reducing interactions between nucleosomes and DNA (DNA is negative), opening chromatin
102
methylation
can activate or repress transcription, different creates different binding sites for readers
103
acetylation cycle
1. enhancer binding protein binds, interation with activator or co-activator (histone acetyltransferase)
2. transcription activated
3. repressor protein recruits histone acetyltransferase, histones deactylated
4. no transcription
104
histone 3 lysine 4 (H3K4)
present in euchromatin, turns on
105
histone 3 lysine 27 (H3K27)
present in heterochromatin, turns off
106
trithorax complex (TrxG)
main histone acetyltransferase complex, activates genes
107
methyl one that competes with TrxG??
108
pioneer transcription factors
can access their binding sites in heterochromatin, can have an active role in opening heterochromatin
109
histone exchange
removal of parts of the nucleosome or the entire nucleosome, followed by replacement with either newly synthesized histones or different components. This swapping can make DNA more accessible, change chemical nature of the chromatin, aid with DNA repair, etc.
110
addition of methyl groups to cytosines
more than 1/2 of the genes in vertebrate genomes contain short CpG-rich regions known as CpG islands (CGIs), rest of the genome is depleted for CpGs
methylation is conserved across generations added by DNA methyltransferases, removed by ten-eleven translocation (TET) proteins
111
Ten-eleven Translocation (TET) proteins
1. hydroxymethylcytosine, remove part of the methyl group
2. deamination of 5-hydroxy citosine
3. base excision repair
112
methyl binding protien
DNA methylation interacts with chromatin remodeling complexes that methylate/deacetylate/close chromatin
113
mRNA
makes proteins
114
rRNA
decodes tRNAs, forms the peptide bond, structure
115
tRNAs
"the adaptor"
116
telomerase
guides the amplification of telomeric repeats
117
snRNAs
splicing
118
RNA leaders
translation control
119
riboswitches
transcriptional and translational control
120
RNA interference (RNAi)
gene silencing pathways that use RNA to modulate gene expression
121
general mechanisms of gene silencing by RNAi
1. dicer - cuts double stranded RNA into small RNAs of 21-2 bp
2. siRNA loaded into RISC
3. RISC has an endonuclease called argonaute, the siRNA is a guide
4. siRNA guides RISC onto the matching mRNA. Binding of small RNA to mRNA target leads to cleavage of the mRNA of suppression of translation
122
sources of dsRNA
- outside the cell, usually a virus
- the cells genome - bidirectional transcription and miRNA genes
123
miRNA
micro interfering RNAs, target different genes than themselves, imperfect complementarity, post-transcriptional regulation of gene expression
124
siRNAs
defense against viruses, transposon repression, heterochromatin formation
125
miRNA pathyway
- miRNA genes are synthesized by RNA pol II as longer RNAs called pri-miRNAs in the nucleus
- has a start condon, stop codon, 3 --> 5 prime utr, poly A signal
- microprocessor processes pri --> pre (80 bp hairpins)
- dicer processes into the 22 bp biologically active miRNAs in the cytoplasm
- bind to the RNA- inducing silencing complex (RISC) and hybridize to mRNAs that are complementary to the miRNAs. Bind to the 3' UTR of the gene
- 3' UTR determines fate
126
miRNA outcomes
- animal cells - imperfect complementarity to mRNA --> translational inhibition, instability (removal of poly A), or sequestration of mRNA into organelles for later use
- plant cells - perfect complementarity, cleavage of mRNA
127
seed region
binding region of the miRNA consists of nucleotides 2 through 8 of the ~22 bp miRNA, the nucleotides of the seed region bind to the 3' UTP of an mRNA
128
inhibition of translation by miRNAs
1. inhibiting initiation complex in ribosome
2. halting translation, ribosome can stay on or fall off
3. inducing mRNA degradation by removing polyA tail
4. sequester mRNAs in p bodies - can be released to the translation machinery or degraded by exo/endo nucleases
129
miRNA genes
hundreds of miRNA genes 1/3 are organized into clusters that are transcribed into a single transcript, which is later processed to form several miRNAs. 1/4 of all miRNAs are processed form transcripts derived form spliced introns
and bind to many 3' UTRs
130
miRNAs and disease
miRNAs are essential for all diseases, using as a new therapeutic
131
siRNAs induce transcirptional gene silencing
siRNAs can induces silencing by going back into the nucleus, recruit proteins that then recruit deacetylases, add DNA methylation --> targets chromatin modifying complexes --> constitutive gene silencing
132
long-non coding RNAs (lncRNAs)
transcribed but not made into proteins, not much is known about them, seem to have key roles in chromatin function and nuclear organization but theris function is still generally not understood. found in complexes with many protein partners, both repressive and activating
133
Xist
Xist from the X chromosome that will become inactivated
Xist and Tsix are antisense to each other
134
mechanisms of X-inactivation
dosage compensation - only on X can be active in any given cell. region on the x chromosome called the x inactivation center, transcribes xist, coats chromosome, serves as a scaffold for proteins that condense and silence the chromatin
135
Tsix
if both tsix and xist are made, tsix binds to xist and prevents it from coating the chromosome
136
chromosome territories
chromosomes are non-randomly arranged in the nuclear space, with many genes occupying preferred positions relative to other regions in the genome or to nuclear structures. These change in different cell types and during development, aging, etc.
137
principles of genome orginization
chromatin domain folding is determined by transcriptional activity of genome regions. Boundaries form at the interface of active and inactive parts. Active regions of different chromosomes are close to one another. higher-order domains of similar activity status. multiple, functionally similar genome domains
138
silenced gene territories
close to nuclear lamina
139
active gene territories
interior of the nucleus
140
nucleolus
rRNA genes
141
nuclear speckles
splicing
142
genome topography and disease
The activity of genes early during development drives nuclear structure. In turn, the topology of the genome play a reinforcing role in maintaining patterns of activity. These are thought to be heritable through meiosis, but are not completely deterministic because they may be able to change
143
Hutchinson-Gilford progeria syndrome
mutations in lamin A genes, age really fast
144
position effect variegation
variegation caused by the inactivation of a gene in some cells through its abnormal juxtaposition with heterochromatin. Spreading meditated by HP-1 and H3 lysine 9 methylation
145
suppression of position affect variegation
mutations in HP1 or H3K9 -losens, expressed
146
Su(var) mutations
block efficient formation of heterochromatin and leave most cells with active w+ transcription
147
E(var) mutations
enhance heterochromatin formation and restrict w+ expression to small patches.
148
barrier insulators
protects a eukaryotic compartment from a heterochromatic compartment
149
homeotic genes
crucial to determine proper body plan during development
150
genomic imprinting
paternal imprinting - paternal allele is silenced
maternal imprinting - maternal allele is silenced
151
imprinting control regions (ICRs)
differentially methylated regions on the paternal and maternal alleles
152
IGF2
on in the paternal chromosome, drives growth
153
H19
on in the maternal chromosome, recruits an insulator protein that blocks IGF2 expression
154
IGF2/Air locus
AIr recruits H3K9 histone methyltransferase G9a and represses chromatin in these regions
154
IGF2/Air locus
AIr recruits H3K9 histone methyltransferase G9a and represses chromatin in these regions
155
transposons
pieces of DNA that can change their position in the genome, may or may not encode for proteins involved in its movement
156
Barbara McClintock
discovered transposons, studied Ac/Dc locus in maize
157
retrotransposons
copy and paste
158
DNA transposon
cut and paste
159
piRNAs
target transposons, piRNA clusters are graveyards of dead transposons that are transcriped and processed into tiny RNAs that then target the transposon mRNA for destruction an its genomic locus for silencing long term. recruits proteins to deacetylase and methylate
