Molecular and Mendelian Genetics Exam 3

regulated transcription

Genes have a basal transcription rate that is modified (regulated) under certain conditions

Constitutive transcription

when a gene is transcribed at constant levels under all conditions

basal transcription

what is the transcription rate of the gene when the transcription factor is not bound

transcription factor

binds to a DNA sequence present near a gene’s promoter; regulated the recruitment of RNA polymerase to the promoter. Two types (Transcriptional activator and transcriptional repressor)

transcriptional activator

Transcription factor that increases gene expression when bound near a gene’s promoter

transcriptional repressor

transcription factor that decreases gene expression when bound near a gene’s promoter

protein domain

functionally independent parts of a protein

transcription factor regulatory domain

part of the protein that regulates transcription

transcription factor binding domain

binds DNA in a sequence-specific manner

allosteric domain

a domain that undergoes an allosteric shift (conformational changes)n when bound by a small molecule effector.

operon

cluster of co-regulated genes, in which multiple coding sequences are co-transcribed from a single promoter with a single regulatory region

activator binding site

binding site for transcriptional activator

operator

binding site for transcriptional repressor

small molecule effector

signaling compounds that control gene expression

negatively inducible transcriptional regulation

binding of a small molecule effector to the transcriptional repressor causes and allosteric shift such that it loses affinity for DNA, and transcription can occur

negatively repressible transcriptional regulation

when the effector is absent, the transcriptional repressor loses affinity for DNA and transcription can occur

positively inducible transcription

when the effector is absent, activator protein binding cannot occur and transcription cannot occur

positively repressible transcriptional regulation

binding of an inhibitor to activator protein prevents activator binding and transcription

structural genes

each coding sequence in an operon

cis-regulatory region

transcription factor binding site near a gene’s promoter

polycistronic transcript

contains multiple coding sequences within a single mRNA, which each coding sequence being proceeded by its own shine-dalgarno sequence, allowing independent initiation of translation of each protein

lac operon

genes that regulate lactose metabolism

Lac Y

encodes lactose permease

Y- mutation leads to no functional permease

lactose permease

encodes by LacY; transmembrane channel that allows lactose to enter the membrane

Lac Z

encodes beta-galactosidase

Z- mutation leads to no functional beta-galactosidase

beta-galactosidase

enzyme that converts lactose to either allolactose or to galactose and glucose

Lac I

encodes the lac repressor

l- mutation makes repressor protein unable to bind to operator; recessive to LacI+

I^s mutation leads to a so-called super repressor; unable to bind to the inducer (allolactose), blocking all transcription; dominant to LacI+

Lac repressor

encoded by lac I; binds to the lac operator and blocks transcription

Lac P

Lac operon promoter, binds protein to block transcription of operon genes; regulatory sequence

P- mutation leads to failing to bind RNA Pol, or doing so weakly

Lac o

lac operator, binds to RNA Pol; regulatory sequence

O- mutation leads to a failure in binding the repressor, resulting in continuous trascription

allolactose

functions as a small molecule effector in the Lac Operon; a lactose isomer

Positive regulation of the Lac Operon

CAP binds and activates transcription; lac operon is transcribed when glucose is low

CAP

a transcriptional activator that binds to the CAP-cAMP binding site upstream of the promoter and bends DNA< increasing accessibility of DNA to RNA Pol

cAMP

a small molecule effector that binds CAP, and allows CAP to bind DNA; cAMP is high when glucose are low

negative regulation in the lac operon

The Lac repressor binds to lacO and represses transcription; when allolactose is present, it binds the lac repressor, leading to an allosteric shift and loss of DNA binding ability

phenotypes when structural genes mutate

phenotypes that affect the presence of one or more gene products, but do not typically affect how an operon is regulated unless their activity is needed for operon regulation

phenotypes when regulatory regions mutate

phenotypes that alter how an operon is regulated; typically, all structural genes are affected in the same way by a given regulatory mutation.

LacA

encodes transacetylase

A- mutation leads to no transacetylase

transacetylase

protects against harmful by-products of lactose metabolism

two classes of regulatory mutations

1. mutations in protein-coding genes where the protein product is involved in operon regulation (i.e the Lac Repressor)

2. mutations in cis regulatory elements within the operon itself (i.e binding sites for the Lac Repressor, CAP, or RNA Polymerase)

plasmid

additional DNA carried on a smaller molecule

partial diploid bacteria

a bacterial cell that temporarily carries two copies of a gene, one on the chromosome and one on a plasmid

mutations in cis regulatory regions

show cis-dominance in partial diploid experiments

in the Lac Operon, any allele of LacO located in cis to the structural gene is dominant to an allele located in trans (not physically connected to the structural gene)

Trp operon

encodes enzymes necessary for tryptophan synthesis in E. coli when tryptophan levels are low; shows a negatively repressible mode of transcriptional regulation

trpR- mutation leads to Trp not fully re-repressing Trp operon transcription

regulated by both transcriptional regulation and attenuation

Trp repressor protein

binds the Trp operator and blocks RNA polymerase binding from the promoter, but only in the presence of co-repressor Tryptophan

attenuation

early termination of transcription, before structural genes of an operon are transcribed; like a dimmer

trpL

leader region of the operon; accomplishes attenuation

contains a short coding sequence that includes 2 Trp codons, and four regions of sequence complementary that can form RNA secondary structure

TrpL mRNA regions

regions 1-4; can fold into multiple possible secondary structures

regions 1-4 secondary structures

most common:

2+3 stem loop, transcription does not attenuate; forms when tryptophan is low

3 + 4 stem loop (intrinsic transcriptional terminator); forms when tryptophan is abundant

differential gene expression

gene expression is regulated differently in different cell types in response to environmental cues, developmental cues, tissue and location specific cues, etc

in eukaryotes, TFs rarely interact with nutrients, environmental cues, etc

steroid hormone signaling

ex: estrogen, cortisol

1. hormone enters its target cell and combines with a receptor protein

2. the hormone/receptor complex binds to a hormone response element in the DNA

3. the bound complex stimulates transcription

4. the transcript is processed and transported to the cytoplasm

5. the mRNA is translated into proteins

peptide hormone signaling

ex: insulin, growth hormone

1. the hormone binds to a receptor protein in the membrane of its target cell

2. the hormone/receptor complex activates a cytoplasmic protein

3. the activated cytoplasmic protein transduces a signal to the nucleus

4. the signal induces a transcription factor to bind to DNA

5. the bound TF stimulates transcription

6. the transcript is processed and transported to the cytoplasm

7. the mRNA is translated into proteins

paracrine signal transduction

short-range signal transduction

juxtacrine signal transduction

adjacent-cell signal transduction

transcription regulation in eukaryotes

1. basal TFs are not sufficient for transcriptional activation

2. “specific” TFs bind at additional cis-regulatory elements (enhancers) to activate/repress transcription of specific genes or sets of genes

activation/repression domain

usually binds other proteins but not RNA Pol directly (in euk)

DNA binding site

short (5-10bp) sequence; often repeated, bound by multiple copies

enhancers

cis-regulatory regions outside of the promoter where transcription factor binding sites are located

key properties of enhancers

• contain binding sites for TFs

• typically have combinatorial control

• are often modular

• may be very distant from the promoter they regulate

  • can be upstream or downstream of transcription start site, and often function normally if moved or inverted into reverse orientation

combinatorial control

multiple TFs regulating one enhancer; enhancers typically contain binding sites for multiple TFs, and its the combo of those TFs that determines whether an enhancer is active

permits fine regulation of spatial and temporal expression patterns of regulated genes

modular

one gene can have many enhancers/ cis-regulatory elements; different enhancers regulate different aspects of spatial/temporal gene expression; mutations in one enhancer don’t affect the activity of other enhancers

permits independent evolution of different aspects of a gene’s expression pattern

methods for detecting and studying enhancers

reporter transgenes and ChIP-Seq

reporter transgenes technique

1. introduce DNA is injected into the nucleus of a zygote and integrates in a random position in the zygotic genome

2. the zygote is implanted in a pregnant female, resulting in each offspring having an independent transgene insertion position

3. each offspring is examined for the presence of the injected DNA to identify transgenic mice

4. each offspring can be bred to generate a transgenic line in which each individual has the same transgene in the same genomic position

chromatin immunoprecipitation (ChIP)

tests binding of a particular protein to chromosomal DNA (chromatin); can be used to assess binding to DNA of interest or the entire genome

steps to ChIP

1. purify chromatin (DNA + bound proteins)

2. add antibody to protein of interest (i.e a TF)

3. precipitate antibody + Protein + DNA using beads coated with an antibody-binding protein

4. purify the immunoprecipitated DNA and characterize it via PCR for a specific sequence (ChIP-PCR) or high throughput DNA sequencing (ChIP-Seq)

ChIP-seq

immunoprecipitated DNA sequences are aligned to a known genome sequence to determine their origin

results are shown as a map of “peaks” corresponding to regions of protein binding

enhancer looping

brings enhancers into close physical proximity to promoters in order to regulate transcription; mediated by molecular bridges

cofactors

components of molecular bridges

insulators

protect regions of chromatin from interacting with each other; when bound to insulator sequences, prevent the action of enhancers on nearby promotes when placed between them

CTCF binding sites are a common type of insulator sequence

topologically associated domains (TADs)

“neighborhoods” that eukaryotic chromatin is organized into; typically, enhancers only loop to promoters within the same TAD; flanked by insulator sequences and bound by insulator proteins

epigenetic modification

a modification to chromatin structure that does not involve changes in the nucleotide sequence; can be transient or stable (inherited from cell to cell/ generation to generation); regulate whether chromatin is euchromatin or heterochromatin; underlie the ability of multicellular organisms to generate multiple differentiated cell types from a single genome

euchromatin

less tightly packaged DNA; associated with trancsribed regions

heterochromatin

>50% of mammalian genome; tightly packaged DNA; associated with transcriptionally silent regions

once established, usually stable in differentiated cells

constitutive heterochromatin

regions of the genome that are always heterochromatic:

regions of repetitive DNA (telomers, centromeres, mobile DNA (retroversion and transposon-derived repeats), inactivated X chromosomes in XX mammals)

Facultative heterochromatin

regions of the genome where chromatin structure varies by cell type and developmental stage

major types of epigenetic modification

chromatin modification (covalent modification of histone tails), chromatin remodeling (moving nucleosomes, changing nucleosome composition), and DNA methylation

acetylation

type of chromatin modificaiton

decreases positive charge of histones, and always leads to a loosening of the nucleosome-DNA interaction

methylation

type of chromatin modification

can increase or decrease nucleosome-DNA interaction depending on the type

histone code

the combination of histone modifications on nucleosomes surrounding a gene may determine the overall effect on transcription

histone mods associated with euchromatin

H3K9ac, H3K27ac, H3K4me

histone mods associated with constitutive heterochromatin

H3K9me

histone mods associated with facultative heterochromatin

H3K27me

writers

histone modifying enzymes that add chromatin modifications

erasers

histone modifying enzymes that remove chromatin modifications

chromatin readers

proteins that bind specific histone modifications and are part of the mechanism that modifies chromatin structure

ChIP-Seq for covalent histone modifications

can reveal active enhancers

chromatin remodeling enzymes

can change the position of nucleosomes, and thus the accessibility of cis-regulatory elements

ex: SWI/SNF proteins

SWI?SNF proteins

one type of remodeling enzymes; can slide nucleosomes along DNA or transfer them to another region of DNA

Open promoters

promoters located in a nucleosome; -depleted region (NDR); many constitutively-expressed genes have them

typically lack TATA boxes and instead have a poly A/T tract located in the NDR

have the histone variant H2A.Z at the +1 nucleosome, an alternative to H2A that makes the nucleosome less stable and more accessible to transcriptional machinery

covered promoters

nucleosomes cover the promoter in cells that do not transcribe that gene; many genes with regulated transcription have these

the binding of transcriptional activators can recruit chromatin remodeling enzymes to displace nucleosome

DNA methylation in mammals

occurs on cytosines on the dinucleotide CG (often written CpG)

catalyzed by enzymes called DNA methyl transferase (DNMTs)

occurs on both strands

methylation association with transcriptional repression

• the inactive x chromosome in female mammals

• regions of mammalian genomes containing repetitive sequences

• near transcription start sites leads to transcriptional silencing

• allele-specific DNA methylation in mammals is responsible for imprinting

imprinting

the expression of a gene is controlled by its parental origin; serves to regulate gene dosage (amt of transcription that can occur from that gene)

CpG Islands

clusters of CpGs occurring throughout the genome in mammals; the methylation state of these affects transcription of associated genes

how DNA methylation regulates chromatin structure

by recruiting histone modifying enzymes

unmethylated CpG Islands association

less histone H1, acetylation of core histones

methylated CpG Islands association

more histone H1, deacetylation of core histones through HDAC recruitment

Igf2

encodes Insulin Growth Factor 2, a paracrine signaling protein

promotes embryonic, fetal, and postnatal growth

too much results in too much growth; occurs when both alleles are paternally imprinted

H19

encodes a non-coding RNA that is processed into several microRNAs

inhibits embryonic and postnatal growth, in part through inhibiting Igf signaling

too much H19 results in too little growth; occurs when both alleles are maternally imprinted

DNAse hypersensitivity

observed in regions of euchromatin; identifies open genomic regions in chromatin that facilitate TF binding to chromatin and induce gene expression

less densely packaged → more accessible → more easily digested by the enzyme DNAse