Module 10 Flashcards
When do supercoils form?
when DNA is underwound or overwound
Relaxed vs strained DNA example
relaxed = 8 turns
strained = 7 turns
Tension leads to . . .
supercoiling
How does supercoiling affect the structure of DNA?
it makes it more compact
the more supercoiled the DNA, the FASTER it will migrate through an agarose gel towards a cathode
What is the role of topoisomerase?
it can relax supercoiled DNA and decatenate interlocked DNA
What does Type I topoisomerase do?
relaxes supercoiled DNA and alleviates the DNA helical constraints
What does Type II topoisomerase do?
unknots & untangles DNA by passing an intact helix through a transient double-stranded break that it generates in a separate helix
Along with Type II topoisomerase, what other protein is involved in the unknotting and untangling of DNA?
DNA gyrase
Briefly describe the structure of topoisomerase (Type II).
- ATPase domain
- cleavage domains
- scaffolding
Describe the structure of a nucleosome.
- 2 loops of DNA wrapped around 8 histones
- when DNA is wrapped around histones it is at its loosest
How is DNA protected within the cell?
by always being bound by proteins
Together, the DNA + proteins are called. . .
chromatin
What are some features of histones?
- small, basic proteins
- 5 major classes
- highly conserved over species
- have amino tails which are highly disordered (protrude from nucleosome)
What do histone modifications affect?
1) the structure + packing of chromatin
2) the access to the DNA of DNA binding proteins
Histones can be. . .
covalently modified
these are proposed to be part of a ‘histone code’ which marks the DNA for specific biological processes
some of the amino acid tails can interact w. the tails of the neighboring nucleosome
Histone acetylation regulates. . .
chromatin condensation (the availability of the DNA for protein binding)
HDAC (histone deacetylase) vs HAT (histone acetyl transferase)
- both are involved in regulation of chromatin structure and gene expression
- HDAC –> condenses chromatin
- HAT –> decondenses chromatin
1st level of DNA compaction
- nucleosomes
- beads on string
- 7 fold compaction
- active DNA ~ 200 nm fiber
2nd level of DNA compaction
- nucleosome + one histone H1 wrapped into another coil
- 100 fold compaction
- inaccessible DNA ~ 30 nm fiber
Where is H1 found?
Histone H1 is located in the interior of the chromatin 30-nm filament
What is a chromosome scaffold?
- can be transcribed
- areas of gene activity are NOT as tightly packed
- high level expression of genes in loop
How does DNA methylation change chromatin structure?
- NO effect on base-pairing
- occurs on CG dinucleotides
- extended regions of CG nucleotides = CpG islands
Where are CpG island found?
usually in PROMOTERS & regulate transcription levels
DNA methylation ______ expression of a gene
REPRESSES
What are de novo DNA methyltransferases (DNMT)?
- enzymes that can methylate CpG islands
- are directed to specific areas of the DNA by DNA-specific binding proteins
How can the methylation of CpG dinucleotides be inherited?
because half-methylated sequences (hemi-methylated) are recognized by maintenance DNA methyltransferases (DNMT genes in mammals)
Describe epigenetic changes
epigenetic changes are reversible and do not change your DNA sequence, but they can change how your body reads a DNA sequence
Are epigenetic changes inherited from cell to cell?
Yes
Examples of epigenetic changes
methylation of cytosines
acetylation of histones in nucleosomes
Epigenetic changes are maintained in the cell but. . .
can be altered by signals (to turn genes on and off)
Why is tight regulation of gene expression important for prokaryotes?
it ensures that a cell’s resources are not WASTED making proteins that the cell does not need at the time
conserves energy + space
What is an operon?
a cluster of genes that are transcribed tog to give a single messenger (mRNA) molecule, which therefore encodes multiple proteins
Describe the Trp operon.
- 5 genes in operon
- each gene in the mRNA has its START & STOP codon
- the promoter is converted into mRNA which is then converted into an inactive protein
What does the Trp operon produce?
produces tryptophan when there is none in the environment the E coli is growing in
Tryptophan absent –>
repressor INACTIVE
operon ON
Tryptophan present –>
repressor ACTIVE
operon OFF
As tryptophan accumulates, . . .
it inhibits its own production by activation the repressor protein, which binds to the operator, blocking transcription
TrpR produces what?
it produces a repressor protein that is initially inactive
it only becomes activated (to repress) once tryptophan binds to it
this allows it to bind to the operator and block transcription
Lactose absent –>
repressor active
operon off
lactose present –>
repressor inactive
operon ON
How does the lac repressor work?
the lac repressor is innately ACTIVE, & in the absence of lactose is switches OFF the operon by binding to the operator
What is the function of allolactose?
allolactose is an isomer of lactose that depresses the operon by inactivating the repressor
bind to the active repressor and forms an inactive repressor
- the lac operon can now be transcribed
trp =
allolactose =
trp = corepressor
allolactose = inducer
both however are NEGATIVE regulators
What are the 3 proteins produced from the lac operon?
lac Z = B-galactosidase
lac Y = permease
lac A = transacetylase
What are some features of the lac operon?
- 3 operators
- all operators are palindromes
- a tetramer binds to the 2 operators (one dimer per operator)
- forms a loop in the DNA b/w it
What is meant by synergistic?
multiple activators cooperate to produce a greater effect
Explain the combinatorial control of the lac operon in response to lactose + glucose
- the ability of the polymerase will depend on the interactions that occur in the promoter region of the DNA
- there are usually several important regulators of a single gene
messages are integrated for a single response
When glucose is LOW –
ATP is LOW, cAMP is HIGH
CAP is activated
transcription
When glucose is HIGH –
cAMP is LOW
CAP remains inactive
no transcription
If lactose is present and CAP is inactive (no cAMP). . .
we will have only very low levels of gene expression
What is CAP?
activator
(positive regulation)
cAMP is a signal for. . .
LOW GLUCOSE
it is a modification of ATP that accumulate in LOW glucose
To be as efficient as possible, E coli should express the lac operon when 2 conditions are met
1) lactose IS available
AND
2) glucose is NOT available
Where does CAP bind?
binds to a region of DNA just before the lac operon promoter and helps RNA polymerase attach to the promoter, driving HIGH levels of transcription
Why is this combinatorial control of the lac operon useful?
it ensures that bacteria only turn on the lac operon and start using lactose AFTER they have used up all of the preferred energy source (glucose)
Glucose high, cAMP low, lactose absent
NO GENE EXPRESSION
Glucose low, cAMP high, lactose absent
NO GENE EXPRESSION
Glucose high, cAMP low, lactose present
LOW GENE EXPRESSION
Glucose low, cAMP low, lactose present
HIGH GENE EXPRESSION
How does methylation of DNA and histones affect the organization of nucleosomes?
causes nucleosomes to pack tightly together
transcription factors cannot bind the DNA & genes are NOT expressed
How does histone acetylation affect the organization of nucleosomes?
results in loose packing of nucleosomes
transcription factors can bind the DNA & genes are expressed
What are some differences in regulation of transcription off prokaryotes vs eukaryotes
- separation of transcription + translation
- separation can BLOCK RNA pol access
- basal transcription is LOW
- majority of regulation is POSITIVE not negative
- more transcriptional regulation + combinatorial control
What is a regulatory element?
the DNA sequence that transcription factors bind to
Where do repressors bind?
bind to silencers
Where do activators bind?
bind to enhancers or enhancer elements
What do mediators do?
- they ‘mediate’ between the activators and general transcription factors
- coactivator
- facilitates binding of the TBP
Where are regulatory sequences found?
- upstream of gene
- downstream of gene
- introns
How does a DNA binding protein recognize specific DNA sequences?
- the major groove gives the necessary info for transcription factors
- major groove gives more info about bases
dif combinations of H bond acceptor, H bond donor, other H, methyl group (code)
Example of DNA binding domain
Helix-loop-helix
What is a DNA binding domain?
- transcription factors are usually made up of several domains
- there is only a small number of possible DNA binding domains
- most transcription factors exist in large gene families
Features of helix-loop-helix
- operates as a dimer
- has 2 components: recognition helix & dimer-forming helix
What is Max?
the human transcription factor which has a HLH structure
- involved in regulating the genes for cell growth and division
What are some features of transcription factors?
1) modular
2) most are in large gene families
3) members of the transcription factor families can homo-dimerise OR sometimes hetero-dimerse
4) combinatorial control
Myc-max
- common in cancer cells
- when it binds to DNA, it interacts with a specific set of proteins and causes ACETYLATION
–> ACTIVATION
Mad-max
- when it binds to DNA, it interacts w a specific set of proteins and causes DEACETYLATION
- REPRESSION
- can no longer divide / grow, but only differentiates
Why is alternative splicing important?
- it allows several proteins to be produced from each gene
- some exons can be spliced OUT along w introns, which increases variety
How does alternative splicing occur?
- splicing REPRESSOR binds to RNA and makes a particular intron-exon boundary harder for the spliceosome machinery to recognize
- won’t recognize that exon - will skip it completely
What are splicing activators?
the splice boundaries b/w the exons and introns may not be v efficient
however, there may be splicing ENHANCERS that bind to RNA and make it more likely that the spliceosome will splice these boundaries
