Exam 3 Flashcards
Chromosomes have a versatile, modular structure for packaging DNA that supports flexibility of
form and function
__ is the generic term for any complex of DNA and protein found in a nucleus of a cell
chromatin
__ are the separate pieces of chromatin that behave as a unit during cell division
chromosomes
Chromatin is 1/3 __, 1/3 __, and 1/3 __
DNA, histones, nonhistone proteins
DNA interaction with __ and __ proteins produces sufficient level of compaction to fit into a cell nucleus
histones; nonhistone proteins
What are histones?
proteins that interact directly with DNA
How do histones interact with DNA?
histones neutralize DNA in the first level of compaction
The core histone complex makes up the
nucleosome
What are the five types of histones?
H1, H2A, H2B, H3, and H4
Of the five types of histones, which ones are core histones?
H2A, H2B, H3, and H4
160 base pairs of DNA wraps twice around a
nucleosome core
40 base pairs of linker DNA connect
adjacent nucleosomes
Which histone associates with linker DNA as it enters and leaves the nucleosome core?
H1
Diameter of DNA helix is
20 A
Diameter of nucleosome core is
100 A
Histones make up __ of all chromatin protein by weight
half
There are about 200-200,000 molecules of each kind of __ protein in chromatin
nonhistone
What are the functions of nonhistone proteins?
- structural role: chromosome scaffold
- chromosome replication: e.g. DNA polymerases
- chromosome segregation: e.g. kinetochore proteins
- transcription: largest group
The __ is the fundamental unit of chromosomal packaging
nucleosome
When DNA wraps twice around nucleosome core octamer, what does that result in?
a 7-fold compaction of DNA
How does spacing and structure of nucleosomes affect genetic function?
- determines whether DNA between nucleosomes is accessible for proteins to initiate transcription, replication, and further compaction
- arrangement along chromatin is highly defined and transmitted from parent to daughter cells during DNA replication
DNA must be condensed __ 7-fold
more than
What does the nucleosome do?
condenses naked DNA 7-fold to a 100 A fiber
What does supercoiling do?
causes additional 6-fold compaction of DNA, achieving a 40-50-fold condensation relative to naked DNA
What does the radical loop-scaffold do?
through progressive compaction of 300 A fiber, condenses DNA to rod-like mitotic chromosome that is 10,000 times more compact than naked DNA
100 A fiber is compacted into 300 A fiber by
supercoiling
What does the radical loop-scaffold model for higher levels of compaction state?
- several nonhistone proteins (NHPs) bind to chromatin every 60-100 kb and tether the 300 A fiber into structural loops
- other NHPs gather several loops together into daisy like rosettes
What is heterochromatin?
chromatin that is highly condensed, and usually inactive transcriptionally. Genes near heterochromatin have reduced expression or are “silenced”
When heterochromatin is constitutive what does that mean?
chromatin is condensed in all cells (e.g. most of the Y chromosome and all pericentromeric)
When heterochromatin is facultative what does that mean?
chromatin is condensed in only some cells and relaxed in other cells (e.g. position effect variegation, X chromosome in female mammals)
What is euchromatin and what is found in it?
relaxed chromatin that is usually transcriptionally active; housekeeping genes are found in this region (e.g. proteins that maintain cell function and are always expressed)
Transcription is controlled by
chromatin structure and nucleosome position
The more compacted DNA is,
the less transcription takes place
What are the three major mechanisms that can regulate chromatin patterns?
- histone modifications
- remodeling complexes
- histone variants
What are histone modifications?
the addition of methyl or acetyl groups
What do remodeling complexes do and how do they do it?
remodeling complexes can alter nucleosome patterns; they do it by
- changing accessibility of promoter sequences
- remove or reposition promoter-blocking nucleosomes
What can histone variants do?
they can cause different nucleosomal structures (e.g. CENP-A at centromeres)
Promoters of transcribed genes are located in
nucleosome free regions
Promoters of non-transcribed genes are wrapped in
nucleosomes
Origins of replication are also
devoid of nucleosomes
When transcription is required, promoters are exposed by
removing or repositioning nucleosomes
In histone modification and chromatin remodeling, the histone tails can undergo __ __ with chemical groups
covalent modification
In histone modification and chromatin remodeling, enzymes can add
chemical groups (methyl groups, phosphate groups, ubiquitin, etc.)
In histone modification and chromatin remodeling, modified tails can alter __ and bind __ __ __
nucleosomes; chromatin modifier proteins
In histone modification and chromatin remodeling, what does acetylation of lysines do?
- prevents close packing of nucleosomes
- favors expression of genes in euchromatin
- de-acetylation results in reduced transcription
In histone modification and chromatin remodeling, what does methylation of lysines and arginines do?
- can either close or open chromatin, depending on specific amino acid modified
- ex: adding methyl group to H3 lysine 9 favors heterochromatin formation
- de-methylation reverses
What is the rate of DNA synthesis in human cells?
about 50 nt/sec
Most mammalian cells have about __ origins
10,000
The human genome has about __ base pairs
3.2 billion (avg 70 million per chromosome)
It would take __ hours to replicate the human genome if there was only one origin of replication
800
T or F? Many origins are active at the same time
T
Nucleosomes are __ and __ during DNA replication
disassembled; reformed
DNA is packaged in nucleosomes within __ of synthesis
minutes
Chromatin fiber unwinds __ to synthesis
prior
Synthesis of histones (in cytoplasm) and transport into nucleus is tightly correlated with
synthesis of DNA
Newly synthesized DNA associates with
new histones
In very early embryo, both __ __ are active
X chromosomes
In humans, random X-inactivation occurs about __ __ after fertilization
2 weeks
Some cells have __ X inactivated, other cells have __ X inactivated
maternal; paternal
All cell descendants have the same
inactive X
Adult female calico cates are __ at X-linked genes
mosaic
In female calico cats heterozygous for X linked mutation:
- some cells have wild-type allele inactivated
- some cells have mutant allele inactivated
What is an example of facultative heterochromatin?
- dosage compensation in mammals so that X-linked genes in XX and XY individuals are expressed at same level
- random inactivation of all except one X chromosome in XX
What are Barr bodies?
darkly stained heterochromatin masses observed in somatic cells at interphase
An XX person has how many Barr bodies?
one
An XXX person has how many Barr bodies?
two
An XXY person has how many Barr bodies?
one
Chromosomes support the __, __, __, and __ of genetic info
packaging, replication, segregation, expression
What are chromosomal abnormalities characterized by a change in the number of chromosomes?
- aberrant euploidy
- aneuploidy
What are chromosomal abnormalities characterized by a change in the structure of chromosomes?
- deletion
- duplication
- translocation
- inversion
Chromosomes have distinct “banding patterns” from staining that can be used as
physical markers for locations of genes
On a chromosome, the short arm is called the __ arm
p
On a chromosome, the long arm is called the __ arm
q
What are the types of chromosomal rearrangements?
- deletion
- duplication
- inversion
- translocation
What is deletion?
the loss of a segment of a chromosome
What is duplication?
the gain of a segment of a chromosome
What is inversion?
the reversal of a region of a chromosome
What is translocation?
the movement of a segment of a chromosome among chromosomes
What is ploidy?
the basic number of chromosomes sets
What is euploidy?
the normal number of chromosomes within a cell for a species
-for ex., the euploid number of chromosomes in a human somatic cell is 46
What does haploid (n) mean? and what is an example of a type of cell that is haploid?
one chromosome set; this is the normal state for some cell type/organisms
-ex: human germ cells
What does diploid (2n) mean? and what is an example of a type of cell that is diploid?
two of the same chromosome set; this is the normal state for many organisms
-ex: human somatic cells
What does polyploid (>2n) mean?
more than two sets of chromosomes
What is transposition?
a type of sequence rearrangement with a significant genomic impact
What are transposable elements?
small segments of DNA that move from one position of DNA to another
Who discovered transposable elements?
Barbara McClintock with her study of mottling of corn color
What do retrotransposons do?
transpose (move their DNA) via reverse transcription of an RNA intermediate
What do transposons (a.k.a DNA transposons) do?
move their DNA directly without an RNA intermediate
What is a common mechanism retrotransposons use?
transcription by RNA polymerase into an RNA that encodes a reverse transcriptase-like enzyme.
The transcriptase-like enzyme can
copy RNA into a single strand of cDNA and then use that single DNA strand as a template for producing double stranded cDNA
Some retrotransposons have a __ __ at the 3’ end of the RNA-like DNA strand, which is similar to mRNA molecules
poly-A
What are the retrotransposons in humans?
- LINES (long interspersed elements
- SINES (short interspersed elements)
What is the hallmark of DNA transposons?
that their ends are inverted repeats (mirror images) of each other
-these repeats are 10-200 bp long
DNA between the transposon’s inverted repeats commonly contains a gene encoding __, a protein that catalyzes transposition through its recognition of those repeats
transposase
What is the DNA transposon mechanism?
- excision of the transposon from its original genomic position
- integration into a new location
- the double-stranded break at the transposon’s excision site is either
- repaired accurately
- the transposon will be lost from the original genomic site after transposition
__% of the human genome consists of transposable elements
44%
Approximately 90% of the transposable elements in the human genome are
retrotransposons
Of the 90% of the transposable elements in the human genome that are retrotransposons, 20% are __ and 13% are __
LINES; SINES
__% of the human genome consists of DNA transposons
3
Most of the transposable elements in the human genome are __ and cannot __
defective; move anymore
Insertion of a transposable element near or within a gene can affect __ and change __
expression; phenotype
Retrotransposon insertion mutations have been shown to cause about 100 know human diseases, including,
forms of hemophilia A, hemophilia B, cystic fibrosis, and muscular dystrophy
What is aneuploidy?
the loss or gain of one or more chromosomes
What are aneuploids?
individuals whose chromosome number is not an exact multiple of the haploid number (n) for that species
__ for any autosome is generally lethal
monosomy
__ for most autosomes is usually lethal, with a few exceptions
trisomy
Most organisms tolerate aneuploidy for
sex chromosomes
What are monosomic individuals?
individuals that lack one chromosome from the normal haploid number (2n-1)
What are trisomic individuals?
individuals that have one chromosome in addition to the normal diploid number (2n+1)
What are tetrasomic individuals?
organisms with four copies of a particular chromosome (2n+2)
How does aneuploidy occur?
chromosomal nondisjunction in meiosis
What is chromosomal nondisjunction in meiosis?
a process by which chromosomes or chromatids fail to separate during meiosis that results in gametes with an abnormal number of chromosomes
Chromosomal nondisjunction in meiosis usually results in the addition of loss of a __ chromosome
single
-resulting organism will have either 45 (one less) or 47 (one more) chromosome in its cells
T or F? Nondisjunction can occur during meiosis I or meiosis II
T
What happens in nondisjunction during meiosis I?
homologous pairs fail to separate during anaphase
What happens in nondisjunction during meiosis II?
sister chromatids fail to separate during anaphase
What are syntenic blocks?
colored segments that contain at least two genes whose order is conserved in the mouse genome
The human genome has about __ genes
25,000
The part of the genome corresponding to exons is the
exome
Most of a genome is non-coding DNA, what is it made of?
- exome (expressed regions) = about 2%
- introns
- centromeres, telomeres, transposable elements
- simple repeating sequences
What are gene-rich regions?
chromosomal regions that have many more genes than expected from average gene density over entire genome -ex in humans: class III region of major histocompatibility complex (60 genes within 700 kb region)
What are gene deserts?
regions of >1 Mb that have no identifiable genes
-3% of human genomes is comprised of gene deserts
T or F? Biological significance of gene-rich regions and gene deserts is not known
T
Exons often encode __ __
protein domains (sequence of amino acids that fold into functional units)
Shuffling, addition, and deletion of domain regions can produce new __ in cells and organisms
functions
Reorganization of domain provides raw material for
evolution
After exon shuffling, protein products have novel
domain architectures
Gene families can evolve by __ followed by __
duplication; divergence
What are gene families?
groups of genes that are closely related in sequence and function
-ex: hemoglobin genes (alpha and beta globes), immunoglobins (antibodies)
Changes in number and arrangement of exons can alter
functions
Duplication and divergence of genes can create genes with both __ and __ functions
new; old
Rearrangements and duplications create many possibilities for
novel functions
Virtually all knowledge of gene structure, expression, and regulation came from studies of
bacteria and bacteriophages
The advent of recombinant DNA technology depended on understanding of
bacterial genes, chromosomes, and restriction enzymes
All bacteria are __, which lack a defined nuclear membrane
prokaryotes
All bacteria lack
membrane-bound organelles
Most bacteria have a cell wall made of __ that surrounds the cell membrane
carbohydrate and peptide polymers
Bacteria have a single
chromosome
Bacteria divide __
rapidly (1 hour in minimal medium, 20 min. in high nutrient conditions)
__ is the most studied and best understood bacterial species
E.coli
E.coli inhabits the intestines of
warm-blooded animals
E.coli can grow in
complete absence of oxygen or in air
E.coli are phototrophic, meaning
they can grow in minimal media
- single carbon and energy source (e.g. glucose)
- inorganic salts
The E.coli genome is tightly packed with
genes
Describe the genome of the K12 strain of E.coli that was sequenced
- 4.6 Mb
- about 90% of genome encodes protein
- 4288 genes, but function known for only 60%
- on average, 1 gene per kb
- no introns
- very little repetitive DNA
- small intergenic regions
Individual E.coli strains contain a subset of the E.coli __
pangenome
What is the core genome of E.coli?
about 1000 genes that are found in all strains
What is the pangenome of E.coli?
the core genome plus all genes that are found in some strains but not others (about 15,000 genes)
The typical bacterial genome is composed of one circular __
chromosome
In bacteria, the DNA molecule condenses by
supercoiling and looping
Each bacterium replicates and then divides by __ __ into two daughter cells
binary fission
__ __ elements dot the genomes of many types of bacteria
insertion sequences (IS)
What are insertion sequences and what do they do in bacteria?
small transposable elements
- inverted repeats (IRs) at ends
- carry transposase gene, which initiates transposition by recognizing IRs
- can move to other locations in genome
- can disrupt genes by insertion into coding regions (cause of many spontaneous mutations)
Tn elements in bacteria are __ __ __
composite transposable elements
Tn elements contain
transposase gene and genes conferring resistance to antibiotics or toxic metals
What are plasmids?
smaller circles of DNA that carry genes beneficial to the host cell
Plasmids don’t carry genes essential to the host, but may
benefit the host under certain conditions
What are some examples of genes that are beneficial to the host
- genes that protect host against toxic chemicals (e.g. mercury) and metabolize environmental pollutants (e.g. toluene, petroleum products)
- pathogenic genes (e.g. toxins produced by S. dysenteriae)
- genes encoding resistance to antibiotics
- multiple antibiotic resistance often due to composite IS/Tn elements on a plasmid
Movement of antibiotic resistance genes TO the plasmid was facilitated by
transposons
Multiple antibiotic resistance genes can be transposed from the plasmid as a
unit
Bacteria must be grown and studied in
cultures
What are some examples of mutant variation in bacteria?
-altered colony morphology
>large or small; shiny or dull; round or irregular
-resistance to bactericides
>antibiotics, bacteriophages (e.g. MRSA!)
-Auxotrophs: unable to reproduce in minimal media
-defective in using complex chemicals from the env
>ex: breaking down lactose into glucose and galactose
-defective in proteins essential for growth
>conditional lethal mutations, e.g. temp-sensitive (ts)
Rapid bacterial multiplication allows for detection of
very rare genetic events
What does effectively haploid mean?
straightforward relationship b/w mutation and phenotypic variation
What happens in selection?
est conditions in which only the desired mutant will grow
What happens in a genetic screening?
examine each colony for a particular phenotype using a technique called replica plating
Genomic analysis has revealed widespread occurrence of __ __ __ in many bacterial species
gene transfer mechanisms
Gene transfer is an important mechanism for __ __ __ __ __ and to development of pathogenic strains of bacteria
rapid adaptation to environmental changes
Describe lateral (or horizontal) gene transfer
traits are introduced from unrelated individuals or from different species
Describe vertical gene transfer
- occurs in sexually reproducing organisms
- traits are transferred from parent to offspring
In the three mechanisms for gene transfer in bacteria,
- donor bacterium provides the DNA that is transferred
- recipient bacterium receives the DNA, which can result in altered phenotype
In conjugation, the F plasmid contains genes for
synthesizing connections between donor and recipient cells
What is conjugation?
direct transfer of DNA from donor cell to connected recipient cell
Donors for conjugation are __
F+ (carry a special F plasmid)
Recipients for conjugation are __
F- (don’t carry an F plasmid)
F plasmid has three
IS elements
__ __ __ cells are formed when an F plasmid integrates into the bacterial chromosome through recombination between IS elements
high frequency recombinant (Hfr)
20-30 different Hfr strains can be generated that differ in the __ and __ of the integrated F plasmids
location; orientation
Integrated F plasmid replicates with __ during cell division
chromosome
Her strains retain all __ __ __ and can be a donor for conjugation with an F- strain
F plasmid functions
Transfer of DNA starts in the F plasmid at the
origin of transfer
Chromosomal genes located next to F plasmid sequences are transferred to the
recipient
Transferred chromosomal DNA recombines into __ __ in recipient
homologous DNA
Usually conjugation terminates before
entire chromosome transfers
How are F’ plasmids created?
- an Hfr plasmid comes out of a bacterial chromosome
- a few chromosomal genes will be removed with it, generating an F’ episome (plasmid)
What are merodiploids?
partial diploids in which two gene copies are identical
What are merodiploids useful for?
complementation testing
To select for Trp+ transformants, plate on minimal media with
histidine and no tryptophan
To select for His+ transformants, plate on minimal media with
tryptophan and no histidine
To screen for His+ Trp+ co-transformants, test Trp+ individual
transformants and His+ individual transformants for growth on
minimal media with
neither tryptophan nor histidine
What are the questions that represent the challenges of gene regulation?
- what should be expressed?
- when should a gene be expressed?
- where should a given gene be expressed?
- how much of a protein is needed, so what level of expression is needed?
RNA polymerase participates in all three phases of
transcription
During initiation, what is present?
core RNA polymerase plus sigma (σ) factor
During elongation, what is present?
core RNA polymerase without σ factor
Most promoters are __ to the transcription start point
upstream
RNA polymerase makes strong contacts at __ and __
-10, -35
Core RNA has four subunits, what are they?
two alpha (α), one beta (β), one beta prime (β')
What happens during initiation?
DNA is unwound and polymerization begins
Elongation continues until
RNA polymerase recognizes termination signal
What are the two kinds of transcription termination in bacteria?
Rho-dependent and Rho-independent
Describe Rho-dependent termination
Rho protein binds to RNA polymerase and removes it from RNA
Describe Rho-independent termination
20 nt sequence in RNA forms stem-loop
What are examples of transcriptional control of gene expression?
- binding of RNA polymerase to promoter
- Shift from initiation to elongation
- Release of mRNA at termination
Binding of RNA polymerase to promoter is the most critical step in
regulation of most prokaryotic genes
What are some examples of post transcriptional control of gene expression?
- stability of mRNA
- efficiency of translation initiation
- stability of polypeptide
Regulation of transcription requires __ that __
regulatory proteins; modify ability of RNA polymerase to recognize and bind promoter
Proteins bind to __ to regulate transcription
DNA
Gene regulation changes with __ context
environmental
Positive regulation __ transcription
enhances
Positive regulatory proteins help __ transcription
activate
Negative regulation __ transcription
inhibits
Negative regulatory proteins help __ transcription
block
Utilization of lactose by E.coli provides a __ __ of gene regulation
model system
What are the two enzymes required for lactose utilization?
permease and β-Galactosidase
What does permease do in lactose utilization?
transports lactose into cell
What does β-Galactosidase do in lactose utilization?
splits lactose into glucose and galactose
In the absence of lactose, both permease and β-Galactosidase are present at __ __ levels
very low
Cells prefer to use __ as an energy source
glucose
In a medium with both lactose and glucose, bacteria will choose __ first, until it’s gone
glucose
Lactose is the __ of the genes encoding permease and β-Galactosidase
inducer
What is induction?
stimulation of synthesis of a specific protein
What is an inducer?
molecule responsible for induction
What are the advantages of using lactose utilization by E.coli as a model for understanding gene regulation?
- Lac- mutants survive and can be maintained on media with glucose and so lac genes are not essential for survival (you can keep your mutants alive)
- simple says for lac expression
- lactose induces a 1000-fold increase in β-Galactosidase activity
- lots of progeny
What does the operon theory state?
one signal can simultaneously regular expression of several clustered genes
Jacques Monod and Francois Jacob hypothesized that lac genes are transcribed together as a single mRNA (polycistronic) from a single
promoter
What are the three structural genes of the lactose operon?
lacZ, lacY, and lacA
What is a promoter?
site to which RNA polymerase binds
What does the cis-acting operator site do?
controls transcription initiation
What does the trans-acting repressor do?
binds to the operator (encoded by lacI gene)
What does an inducer do?
prevents repressor from binding to operator
What is the lac-operon?
a cluster of genes transcribed simultaneously (lacZ, lacY, lacA)
Describe lac-operon induction when lactose is present
- inducer binds repressor
- repressor changes shape and cannot bind to operator
- RNA polymerase binds to the promoter and initiates transcription of the polycistronic lac mRNA
Repression of lac gene expression occurs in the __ of lactose
absence
Lac repressor is a __ regulatory element
negative
lacZ encodes
β-Galactosidase
lacY encodes
permease
lacA encodes
transacetylase
lacA is not necessary for
lactose catabolism
lacZ and lacY mutations led to new phenotype, what is it?
inability to utilize lactose
If cells grow in lactose medium, the mutations
complement
If cells do not grow in lactose medium, mutations
fail to complement and are in the same region
What is characteristic of lacZ-?
- inability to produce β-Galactosidase
- no induction of β-Galactosidase activity by lactose inducer
What is characteristic of lacY-?
- inability to produce permease
- no induction of permease activity by lactose inducer
What is characteristic of lacI-?
constitutive expression of β-Gal and permease
__ __ express the enzymes in the absence and presence of inducer
constitutive mutants
Why must lac I be a repressor?
cells require lac I protein to prevent expression of lac Z and lac Y in absence of an inducer
The PaJaMo experiment provided evidence that lacI encodes a
repressor
Jacob and Monod proposed that lacI encodes a repressor that binds to an operator site near the __ promoter
lac
What does the binding of an inducer to a repressor do?
changes the shape of the repressor so that it can no longer bind to DNA
When there is no inducer present, the repressor is able to
bind to DNA
Repressor is an allosteric protein, meaning that
it undergoes reversible changes in conformation when bound to another molecule
lacI- mutants have a __ __ that cannot bind to operator
mutant repressor
lacI(s) mutants have a __ that binds to operator but can’t bind to the inducer
superrepressor
In lacI(s) mutants, lac genes are __ in the absence and the presence of inducer
repressed
lacO(c) mutants have a mutant operator that can’t bind the __
repressor
In lacO(c) mutants, lac genes are __ in the absence and the presence of inducer
expressed
What are the 2 ways to get constitutive expression?
- mutation in lacI (lacI-) that prevents the repressor from binding the operator whether lactose is present or not
- Mutation in operator DNA sequence (lacO(c)) the prevents repressor from recognizing and binding
How can you distinguish between the two ways to get constitutive expression?
using the cis/trans test with merodiploids
Describe trans-acting elements
can diffuse through the cytoplasm and act at target DNA sites on any DNA molecule in the cell
Describe cis-acting elements
can only influence expression of adjacent genes on the same DNA molecule (operator, promoter, etc)
Cis DNA elements need to be on same chromosome as
genes they regulate
Trans elements are proteins produced on one molecule that can interact with
DNA on either molecule
If lacI(s) is cis-acting, then lacZ+ chromosome will be
inducible
If lacI(s) is trans-acting, then lacZ+ will be
non-inducible
lacI(s) protein acts in
trans
lacO(c) acts in
cis
The lacO(c) mutation affects expression of genes only on
the DNA that it is located on
Why does the O+ operator have no effect in the lacO(c) mutation?
because it is adjacent to a mutant lacZ on the plasmid and can’t impact the lacZ+ on chromosome
Initiation of transcription under control of regulatory genes whose protein products bind to DNA near promoter alter
RNA polymerase
What is the biochemical evidence for lac repressor binding to lacO?
- lac repressor has two separate domains
- lac repressor has a helix-turn-helix (HTH) motif
- most DNA-binding regulatory proteins are oligomeric (one domain), with two to four subunits
Mutated DNA sequences in many different lacI- mutants are clustered in the __ __ domain of lac repressor
DNA-binding
Mutated sequences in many different lacI(S) mutants are clustered in the __ __ domain of the lac repressor
inducer-binding
A protein with an HTH (helix-turn-helix) motif has two __ __ separated by a turn in the protein
α-helical regions
The HTH motif fits into the major groove of
DNA
One of the α-helical regions of a protein with an HTH motif has amino acids that ‘recognize’ a specific
DNA sequence
HTH motifs are found in many __ __ proteins
DNA-binding
lac repressor tetramer binds to __ sites
two
lac repressor is a tetramer, with each subunit containing a DNA-binding __ __
HTH motif
Two repressor subunits of lac repressor bind to
O1
Two repressor subunits of lac repressor bind to either __ or __
O2 or O3
The binding of the repressor subunits of lac repressor to O1, O2, and O3 causes the formation of a loop that leads to
highly efficient repression
When lac repressor is bound to lac operator, functional binding of RNA polymerase to the promoter is
blocked
Many negative regulators (e.g. lac repressor) prevent transcription initiation by blocking
the functional binding of RNA polymerase
Many positive regulators (e.g. CRP-cAMP) establish contact with RNA polymerase that
enhances transcription initiation
The lac operon of E.coli is regulated by both __ and __
lactose; glucose
When both glucose and lactose are present, only __ is utilized
glucose
Lactose induces __ __ __, but only in the absence of glucose, even if lactose is present
lac mRNA expression
Positive regulation increases transcription of lacZ, lacY, and lacA only when
lactose is present and glucose is absent
Lactose prevents repressor from binding to
lacO
lac repressor is a __ regulator of lac transcription
negative
lac mRNA expression cannot be induced if
glucose is present
Glucose controls the levels of
cAMP
cAMP binds to __ __ __
cAMP receptor protein (CRP)
CRP-cAMP is a __ regulator of lac transcription, but ONLY in absence of glucose
positive
Describe catabolite repression
overall effect of glucose is to prevent lac gene expression by limiting availability of cAMP
CRP-binding sites have a two-fold __ __
rotational symmetry
CPR protein binds as a
dimer
CRP-binding site consists of two recognition sequences, one for each
subunit of the CRP dimer
CRP-cAMP complex makes direct contact with
RNA polymerase
Without interaction with CRP-cAMP, RNA polymerase can bind to the promoter but is less likely to __ __ __ __ __
unwind DNA and initiate transcription
What is a reporter gene?
protein-encodind gene whose expression in the cell is quantifiable by sensitive and reliable techniques
How do you measure gene expression?
- fuse coding region of lacZ to cis-acting regulatory regions from other genes
- conditions that induce expression of gene of interest will generate β-gal
How do you control gene expression?
- fuse the lac regulatory sequences to the coding region of a foreign gene
- inducible expression of the foreign gene controlled by IPTG
lacZ fusion is used to perform genetic studies of the regulatory region of
gene X
Conditions that regular expression of the test regions from gene X will alter the levels of
β-galactosidase
Specific regulatory sites can be identified by constructing and testing mutations in the test regions of
gene X
Expression of gene X is under the control of the
lac regulatory system
Expression of human growth hormone in E.coli is controlled by
lac control region
How do eukaryotes use complex sets of interactions?
- regulated interactions of large networks of genes
- each gene has multiple points of regulation
- turning on and off genes in right place ad time
What are the themes of gene regulation in eukaryotes?
- environmental adaptation, growth, and division in prokaryotes
- maintenance of homeostasis in multicellular eukaryotes
- genes are turned on and off in right place and time
- differentiation and precise positioning of tissues and organs during embryonic development
Eukaryotic genomes are __ than prokaryotic genomes
larger
Compared to prokaryotes, eukaryotes have additional
levels of complexity for controlling gene expression
__ __ in eukaryotes makes DNA unavailable to transcription machinery
chromatin structure
Additional __ __ events occur in eukaryotes
RNA processing
In eukaryotes, transcription takes place in __ and translation takes place in __
the nucleus; the cytoplasm
Both eukaryotes and prokaryotes utilize DNA binding proteins for
transcriptional regulation
There are multiple steps where production of the final gene product can be regulated in
eukaryotes
Eukaryotes can regulate these steps to control __ __ in different tissues
cell differentiation
What does RNA polymerase I do?
transcribes genes that are the major RNA components of ribosomes (rRNAs)
What does RNA polymerase II do?
transcribes genes that encode all proteins
What does RNA polymerase III do?
transcribes genes that encode the tRNAs and certain other small RNA molecules
RNA pol II catalyzes synthesis of the __ __, which is complementary to the template strand of the gene
primary transcript
Most RNA pol II transcripts undergo further processing to generate __ __
mature mRNA
What does RNA splicing do?
removes introns
What does addition of 5’ GTP cap do?
protects RNA from degradation
What are the further processes that RNA pol II transcripts must undergo to generate mature mRNA?
- RNA splicing
- addition of 5’ GTP cap
- cleavage of 3’ end by ribonuclease, and addition of 3’ polyA tail (poly-A polymerase)
Promoters are usually adjacent to
the protein-coding gene
Promoters include the transcription initiation site and often have
about 7 base pair TATA box
Binding of RNA pol II allows __ __ of transcription
basal level
Describe enhancers
can be distant (10,000s of bps) from gene, or even within gene introns or reversed in orientation
Binding of proteins can augment or repress __ __
basal transcription
Trans-acting factors interact with cis-acting elements to control rates of
transcription initiation
How do the direct effects of transcription factors come about?
- through binding to DNA
- basal factors
- activators and repressors
How does the indirect effect of transcription factors come about?
through protein-protein interactions
Basal transcription factors assist the binding of RNA pol II to
promoters
What are the key components of the basal factor complex?
- TATA box-binding protein (TBP)
- binds to TATA box
- first of several proteins to assemble at promoter
- TBP-associated factors (TAFs)
- bind to TBP assembled at TATA box
Basal factors bind to promoters of all __ __ __
protein-encoding genes
What is the ordered pathway of assembly at promoter?
- TBP binds to TATA box: produces “bend” in DNA of TATA box
- TAFs bind to TBP
- RNA pol II binds to TAFs
RNA pol II associates with basal complex and initiates
basal level of transcription
Activators are transcription factors that bind to
enhancers
What is the significance of activators?
binding of different activators to enhancers is responsible for much of the variation in levels of transcription of different genes in different cell types
How do activators increase levels of transcription?
by interacting directly or indirectly with basal factors at the promoter
Binding of activators to enhancers increases
transcriptional levels
Low level transcription occurs when only __ __ are bound to promoter
basal factors
When basal factors AND activators are bound to DNA, rate of transcription __
increases
What are the two functional domains of activator proteins?
- sequence-specific DNA binding domain (bind enhancer)
2. transcription-activator domain (which binds other trans-factors)
__ __ leads to physical interaction of distant DNA regions
DNA looping
What are the mechanisms of activator effects of transcription?
- stimulate recruitment of basal factors and RNA pol II to promoters
- recruit coactivators to open chromatin structure
What are the effects of activators on transcriptions?
- stimulate recruitment of basal factors and RNA pol II
- stimulate activity of basal factors
- facilitate changes in chromatin structure
What do the DNA-binding domains of activator proteins do?
interact with major groove of DNA
-certain amino acids have high-affinity binding to specific nucleotide sequence
What are the three best-characterizes motifs?
- helix-loop-helix (HLH)
- helix-turn-helix (HTH)
- zinc finger
__ __ __ are activators, but only in the presence of specific hormones
steroid hormone receptors
Steroid hormones don’t bind directly to DNA but are __ of steroid hormone receptors
coactivators
In the absence of hormone, steroid hormone receptors can’t bind to DNA and can’t
activate transcription
In the presence of hormone, steroid hormone receptors bind to enhancers for specific genes and activate
expression
T or F? Not all transcription factors activate gene expression
T
When a repressor binds to the same enhancer sequence as the activator, what effect does it have on the basal transcription level?
no effect
Describe quenchers
bind to the activator but do not bind to DNA; the repressor blocks the activator from functioning
Some repressors eliminate virtually all basal transcription from a promoter by blocking
promoter access
In humans, about __ genes or __% of genes encode transcriptional regulatory proteins
2000; 10%
Each regulatory protein can act on __ genes
many
Each regulatory protein can have __ of enhancers
dozens
The same transcription factors can be an activator or a repressor depending on
- the cell that it’s in
- which cis-regulatory element it binds to
Regulatory proteins have vast complexity for precise control of
gene expression
What are the two methods that cells use to regulate transcription?
- binding of transcription factors to enhancers
- DNA methylation
What does binding transcription factors to enhancers do?
modulates the spatial and temporal expression of many genes that are expressed only in particular tissues at specific times during development
Describe methylation
(a biochemical modification of DNA itself)
-a methyl (CH3) group is added to the 5th carbon of the cytosine base in a 5’ CpG 3’ dinucleotide pair on one strand of the double helix
DNA methylation is important for controlling expression of
“housekeeping genes”
DNA methylation also regulates some
cell-type specific genes
Why can DNA methylation alter gene expression heritability without changing the base sequence of DNA
because methylation affects transcription levels, and methylation patterns are copied during DNA replication (this is called the epigenetic phenomenon)
Methylation is key to epigenetic in mammals and is called
genomic imprinting
What organisms have no DNA methylation?
C. elegans and yeast
What organisms have very little DNA methylation?
other invertebrates and lower euks
DNA methylation at CpG islands __ gene expression
silences
DNA methylation usually __ the transcription of eukaryotic genes
inhibits
DNA methylation usually inhibits the transcription of eukaryotic genes particularly when it occurs in the vicinity of the
promoter
In vertebrates and plants, CpG islands occur near many
promoters of genes
CpG islands are commonly __ in length and contain a high number of __
1000 to 2000 bp; CpG sites
DNA methylation is thought to play an important role in the silencing of tissue-specific genes to prevent
them from being expressed in the wrong tissue
Nucleosomes can make promoters __
inaccessible
What are epigenetic changes?
changes in chromatin structure that are inherited from one generation to the next
- DNA sequence is not altered
- but particular cells with altered chromatin will have altered gene expression, which can be inherited from one cell generation to the next
Chromatin reduces binding to basal factors and RNA pol II to
very low levels
Nucleosomes can be __ or __ by chromatin remodeling complexes
repositioned or removed
After remodeling, DNA at promoters and enhancers becomes __ __ to transcription factors
more accessible
Transcription is active near __ CpG islands
unmethylated
CpG islands are regions with a high concentration of
CpG dinucleotides
Near genes, CpG islands are usually unmethylated because
an activator binds and blocks access by DNMTs
-the chromatin is open and transcription is activated
DNA methylation at CpG islands __ gene expression
silences
In the absence of activators, the CpG islands become
methylated
Methyl-CpG-binding proteins (MeCPs) binds and close the
chromatin structure
Gene expression repression is often long-term because
the methylation pattern is maintained through numerous cell divisions
Long-term repression through DNA methylation is called
silencing
DNA methylation is an __ __ because it can heritably alter gene expression without changing DNA sequence
epigenetic phenomenon
Cytosine methylation pattern is copied during
DNA replication
DNA methylation patterns are copies during DNA replication by
a special DNMT present at the replication fork
-this DNMT recognizes semi-methylated DNA (on the parent strand) and methylates the newly synthesized strand
Sex-specific DNA methylation is responsible for
genomic imprinting
What is the Mendelian rule?
parental origin of allele does not affect F1 phenotype (usually!)
Describe genomic imprinting
expression of a gene depends on whether it was inherited from the mother or father
-epigenetic effect (no change in DNA sequence)
__ __ __ is transcriptionally silenced if it was transmitted from the father and the maternally inherited allele is expressed
paternally imprinted gene
__ __ __ is transcriptionally silenced if it was transmitted from the mother and the paternally inherited allele is expressed
maternally imprinted gene
imprinted =
silenced
Clinical geneticists realized that genomic imprinting existed long before molecular biology techniques made it possible to confirm its existence, how?
pedigree analysis of rare diseases
- the patterns were clear in certain rare cases where the condition was caused by a deletion that removed the imprinted gene
- the inheritance of the deletion and the disease could be followed in karyotypes
When examining a pedigree chart of an imprinted gene, the sex of the parent carrying a mutant allele determines offspring __, not sex of the offspring!
phenotype
If there is a deletion of a paternally imprinted autosomal gene.. fathers can pass the deletion to their sons and daughters and they will not be affected because
the child’s wild-type maternal allele would be expressed
The genomic imprint is ___ during mitosis
maintained
Patterns of DNA methylation must be __ during meiosis before being passed on to the next generation
reset
Imprinting is
sex-specific
Methylation is removed in
germ-line cells
Imprinting occurs in the __ __ and is accompanied by heavy __
germ line; methylation
Epigenetic imprints are erased during __ __ __ and reset by __ __ __
germ-line development; sex-specific patterns
Complementary base pairing between a small RNA and mRNA can prevent
gene expression
What are the 3 classes of small regulatory RNAs that have been identified
- micro-RNAs (miRNAs)
- small interfering RNAs (siRNAs)
- Piwi-interacting RNAs (piRNAs)
Each small RNA class leads to the production of
single-stranded RNAs of slightly different lengths
Each small RNA class is in the range of __ nucleotides
21-30
What are the targets of miRNAs?
mRNAs
What are the effects of miRNAs?
-block mRNA translocation/ destabilize mRNAs
What are the targets of siRNAs?
mRNAs
What are the effects of siRNAs?
block translation/ destabilize mRNAs
In order to prevent gene expression, small RNAs work with proteins in the __ family
Argonaute
-form ribonucleoprotein complexes
The small RNA in each ribonucleoprotein complex guides the complex to
particular nucleic acid
- this nucleic acid target will have perfect or partial complementarity with the small RNA
- the mRNA will not be expressed in most cases
- this is a type of post transcriptional RNA modulation
