STUDY GUIDE

Gene regulation

complex bc eukaryotes have…

• many diff cell types with diff functions

• DNA packaged into chromatin that must be opened or closed

• multiple steps in gene expression

• many regulatory proteins and DNA elements working together

Where in the central dogma does majority of gene expression take place?

during transcription

Housekeeping gene

always on in all cells bc it’s needed for basic survival

Conditionally expressed gene

only tuned on in certain conditions, times, or cell types

Chromosome territory

each chromosome occupies its own specific area inside the nucleus

Interchromosomal domain

the spaces between chromosome territories where transcription & RNA processing occur

• genes near active regions or near interchromosomal domains are more likely to be transcribed

Chromatin remodeling complex

changes the structure of chromatin so genes can be turned on/off

1. slides nucleosomes

2. remove or reposition nucleosomes

3. replace histones with variants

energy: ATP

PIC (pre-initiation complex)

general transcription factors + RNA poly II

• assembles at the core promotor 

Cis-regulatory element

a DNA sequence near a gene that controls transcription

• promotor

• enhancer

• silencer

Activator protein

protein that binds to an enhancer

What can happen to the PIC when an enhancer is bound?

• PIC can be stabilized/anchored

• PIC can be recruited faster

• transcription rate increases

Activator domains

• DNA-binding domain: binds enhancer

• activation domain: interacts with PIC or remodeling proteins

Repressor protein

protein that binds to a silencer

How does a repressor block transcription?

• blocks activator binding

• prevents PIC assembly

• recruits chromatin compaction proteins

Response element

DNA sequence that responds to signals (like hormones or stress) by allowing specific transcription factors to bind and regulate gene expression

Combinational gene regulation

multiple proteins (activators, repressors, remodelers) work together to control one gene

Mutations can affect cis-regulatory fxn & transcription by…

• preventing binding of activators (↓ transcription)

• preventing repressors (↑ transcription)

• create new binding sites

Reporter assay

• measures gene expression

• attach reporter gene to a promotor or regulatory element. if element is active, the reporter produces a measurable signal

Alternative splicing

different combinations of exons are kept or removed to create different mRNAs from one gene

Splice variant

the different mRNA versions produced

PolyA tail

• protect mRNA from degradation and help with translation

• more tail = more stability & more translation

• short tail = degraded faster

What happens when an mRNA is deadenylated & decapped?

it’s exposed & quickly degraded by exonucleases

Why might a cell not translate an mRNA right away?

• mRNA is stored for later

• speedy response

• localized translation

Post-translational modifications

• like an on-off switch

• adding or removing a group (like phosphate) can turn a protein ON or OFF very quickly

Ubiquitin

targets a protein for degradation

• E3 ubiquitin ligase = protein that adds the ubiquitin groups

• proteasome = protein complex that degrades the protein

Epigenetics

heritable changes in gene expression without changing the DNA sequence

Epigenome

all the epigenetic marks on the DNA and histones in a cell

3 epigenetic mechanisms

• DNA methylation

• histone modification

• noncoding RNAs - RNA interference

DNA methylation

adding a methyl group to DNA to turn genes off

Histone modification

adding/removing chemical groups to histone proteins to loosen or tighten chromatin

Noncoding RNAs

small RNAs bind mRNA to block translation or cause mRNA destruction

Monoallelic expression

only one allele (either the mom or dads copy) is expressed, while the other is silenced by epigenetic marks

Epimutation

an abnormal epigenetic change (like incorrect methylation or histone modification)

• in terms of imprinting: if the only active allele gets silenced by epimutation, no working copy of the gene remains → disease

What residue along the DNA can be methylated?

cytosine (especially in CpG sites)

What is the effect on transcription when methylation occurs?

transcription decreases or stops (gene is silenced)

Histone tail

part of the histone that is modified

Histone acetyltransferases (HATs)

add acetyl groups → open chromatin → increase transcription

Histone deacetylases (HDACs)

remove acetyl groups → tighten chromatin → decrease transcription

When H3K9 is acetylated…

chromatin opens → transcription increases

When H3K9 is deacetylated…

chromatin tightens → transcription decreases

Histone code

the idea that specific patterns of histone modifications act like a “code” that tells the cell whether a gene should be on or off

Types of RNAs that trigger the interference pathway

• miRNA

• siRNA

RISC

• RNA-induced silencing complex

• uses small RNAs to bind and silence target mRNAs (either block translation or degrade mRNA)

How does the trigger RNA guide RISC to the target mRNA?

the small RNA base-pairs with the complementary sequence on the target mRNA

What happens to the target mRNA when RISC is bound?

• it is degraded OR

• translation is blocked

either way → less protein is made

Bisulfite sequencing

• tehcnique used to measure DNA methylation

  • whether certain cytosines in DNA are methylated or not

  • unmethylated cytosines → uracil

  • methylated cytosines → stay same

ChIP

• Chromatin ImmunoPrecipitation

• used to find out which proteins (like transcription factors or modified histones) are physically bound to specific DNA regions in the cell

• cells are lysed & DNA is fragmented into smaller pieces

• protein of interest is purified using an antibody that recognizes the protein

• DNA is isolated & identified 

  • southern blotting

Cross fostering

• pups born to high licking or low licking mothers were swapped within 12 hours of birth and raised by the opposite mother

• found that the methylation pattern of the GR promoter match the rearing mother, not the biological mother

  • pups raised by high-LG mothers had low methylation, even if born to low LG mothers

  • pups raised by low-LG mothers had high methylation, even if born to high-LG mothers

• epigenetics b/c pattern changed based on the environment (maternal care caused epigenetic modifications which then changed gene expression and stress response)

Wildtype genotype

the normal, most common DNA sequence in a population

Wildtype phenotype

the normal physical trait or function seen in most individuals

Mutant genotype

a DNA seqeunce that has changed from the wildtype

Mutant phenotype

a physical trait or function that is different due to the mutation

Adaptive mutation

a mutation that seems to arise when it provides a benefit under stressful conditions

Random mutation

• mutations occur by chance, not because they are needed

• happen before selection

Mutation hot-spot

a region in DNA that mutates more often than others (due to sequence, structure, or repair difficulty)

Founder mutation

a mutation that appears in the DNA of a small founding population and gets passed to many descendants

Sources of spontaneous mutations

• DNA replication errors 

• spontaneous chemical changes

• transposable elements

Replication mistakes

wrong base inserted

Trinucleotide repeat expansion

repeats grow in number, causing gene disruption

Replication slippage

extra or missing bases inserted

Tautomeric shift

temporary base form change → mispairing

DNA replication - mutation

• most mutations occur when DNA is copied

• mistakes during replication that aren’t repaired become permanent mutations

Induced mutations - DNA damage

mutagens damage DNA… if the damage isn’t repaired correctlty, it becomes a mutation

Mutagen

anything that increases the rate of mutation (chemicals, radiation, etc.)

Base modifier

• alkylating agents

• chemical that adds groups to bases → causes incorrect base pairing

Intercalating agent

flat molecule that inserts into DNA → causes frameshift mutations

Ionizing radation

causes DNA damage b/c breaks sugar-phosphate backbone → double-strand breaks

UV light

causes pyrimidine dimers

Pyrimidine dimers

• covalent bonds between adjacent T or C bases → distort DNA

• lead to mutations b/c DNA polymerase may skip, misread, or insert wrong bases → substitutions or frameshifts

Loss-of-function

protein works less or not at all

Null allele

complete loss of protein function

Deleterious/lethal allele

harmful or causes death

Gain-of-function

protein gains a new or too strong activity

Somatic mutation

in body cells → not passed on

Germline mutation

in egg/sperm → can be inherited

Base pair substitutions

one base replaced by another

Insertion/deletion

extra or missing bases

Frameshift

insertion/deletion not in multiples of 3 → shift reading frame

Types of substitutions

• transition: purine ↔ purine (A↔G) or pyrimidine ↔ pyrimidine (C↔T)

• transversions: purine ↔ pyrimidine

Synonymous

same codon meaning

Silent

no change in protein N

Neutral

amino acid change but similar → little/no effect

Missense

one amino acid changed

Nonsense

codon becomes STOP → truncated protein

Intragenic suppressor

second mutation in same gene restores function

Intergenic suppressor

mutation in different gene restores function

Forward mutation

wildtype → mutant

Reverse mutation

mutant → back to wildtype

Revertant

the actual organism where the mutation was corrected

Amino acid labeling

single letter or three letter code + position number

• ex: Arg117 or R117

Mutation labeling

original amino acid + position + new amino acid

• ex: R117H (Arg at position 117 becomes His)

Batten disease

misfolded or missing lysosomal protein → toxic buildup

Sickle cell disease

missense mutation (Glu → Val) → hemoglobin clumps → sickled cells

Cystic fibrosis

CTFR misfolded or missing → mutant phenotype

Transposable element

a DNA sequence that can move (transpose) to new locations in the genome

Transposon

“cut and paste” DNA directly

Retrotransposon

“copy and paste” using RNA → reverse transcription

Transposition

movement of a transposable element to a new DNA site

Replicative transposition

• copy is made and inserted elsewhere → increases genome size

• increases genome C value (copy increases DNA content)

Conservative transposition

element is cut out and moved → genome size same