Genetics (224) Lectures 7 (after slide 16), 8, and 9 (slides 43-53)

Positive control (of transcription)

Involves the binding of an activator protein to a regulatory DNA sequence, initiating transcription (CAP-cAMP complex binding to the CAP site in the lac promoter)

Negative control (of transcription)

Involves the binding of a repressor protein to a regulatory DNA sequence, preventing transcription

Active repressor

Protein that blocks expression of genes in an operon by binding to the operator to prevent transcription

Arabinose operon

• One regulatory (araC) protein is responsible for both positive and negative control of transcription

• It is both inducible and repressible

• Contains 3 structural genes coding for enzymes to metabolize arabinose: araB, araA, araD

• Transcription is controlled by the promoter (P_ara) and 3 operator sites: araI, araO₁, araO₂

What must occur for RNA polymerase to successfully transcribe the arabinose operon?

• The CAP-cAMP complex must bind to araI

• The araC protein must bind to arabinose, and that complex must also bind to araI

• Overall, glucose must be low (causing high cAMP) and arabinose must be present for the operon, containing genes to break down arabinose, to be transcribed

How is the arabinose operon repressed?

When arabinose is absent, araC proteins (which are obviously not bound to ara) bind to both the araI and araO sequences and then link together, creating a loop in the DNA and preventing the binding of RNA polymerase

araC

• The regulatory protein (responsible for both positive and negative regulation) in the arabinose operon. Encoded by the araC gene

• Exists as a homodimer when bound to araI and arabinose to activate transcription

Initiator region (arabinose operon)

• araI

• Is bound to by araC bound to arabinose to activate transcription

• Can also be bound to araC in the absence of arabinose, which binds to another araC bound to araO₂ to repress transcription (makes a loop in the DNA)

• Located between the operator regions and the prooter

Repressible operons

• Operons involved in anabolic pathways

• The end product blocks transcription of the operon

• Ex. trp operon in E. coli

Attenuation

• Mechanism to terminate transcription in bacteria

• Involves 4 repeat DNA sequences in the attenuator region that can bind together to either allow transcription to continue (regions 2+3) or halt it (regions 1+2 and 3+4)

Leader sequence

Ex. trpL: sequence in the trp operon that contains the attenuator region (which is composed of the 4 domains that can form stem-loops structures)

How is it determined whether attenuation will occur in trpL?

The leader sequence codes for a small polypeptide containing 2 tryptophans, so if trp levels are low, the ribosome will stall out on region 1. This leads to domains 2+3 binding together rather than 1+2 and 3+4, and transcription continues so that more trp can be made. If there is enough trp, the entire leader sequence will be translated and the ribosome will fall off when it reaches a termination signal, allowing stem loops between 1+2 and 3+4 to form

Attenuator region

Region in the leader sequence which is composed of the 4 domains (4 repeat DNA sequences that are complementary to each other) that can form stem-loops structures

3-4 stem loop (of mRNA)

• The termination stem loop

• Stops the movement of RNA polymerase along DNA within the leader sequence

• Followed by long chain of uracils (which is what causes the RNA polymerase to fall off)

2-3 stem loop (of mRNA)

• The antitermination stem loop

• Forms when region 1 in the attenuation region isn’t available (when the ribosome stalls because there isn’t enough trp, sitting on region 1)

• Prevents formation of 3-4 stem loop, allows RNA polymerase to continue

cI gene

• Gene in the phage λ genome

• Encodes a repressor that represses lytic growth and promotes lysogeny

cro gene

• Gene in the phage λ genome

• Encodes a repressor that represses lysogeny and promotes lytic growth

Genetic switch

• In general terms, regulatory mechanisms that turn genes on or off in response to environmental signals

• There are two possible states in the λ phage (lysogeny or lytic growth), determined by the interactions of regulatory proteins with promoters

Alternative sigma factors

• Sigma factors that can recognize different promoters from sigma70, the sigma factor that is used most of the time

• You can’t switch out RNA polymerase since bacteria only have one, but you can switch sigma factors

• Used to turn on operons (rapidly)

• Ex. in gram positive bacteria that produce endospores

Regulons

Large sets of genes

General transcription factors

Molecules that help RNA polymerase II bind to the promoter in eukaryotes (ex. TFIIA, TFIIB)

Promoter proximal elements

Regulatory DNA sequences located near the promoter (within 100 bp of the gene) where GTFs bind

Activator proteins

Proteins that bind to enhancer DNA sequences to attract RNA polymerase

Enhancer sequences

• DNA sequences that enhance transcription, and are bound to by activator proteins

• Can be located far away on the strand of DNA but fold to be close to the promoter

Core promoters

• The main important section of the promoter region, where RNA polymerase II and GTFs bind

• Usually includes the TATA box, Inr, FRE, and DPE

• These alone are usually not sufficient for regulated transcription

Coregulators

Proteins that regulate transcription (either enhance or repress it) without binding to DNA directly, but interacting with transcription factors and other proteins

Coactivators (mediator)

Type of coregulator that promotes transcription (ex. the mediator complex)

Enhancer regions/upstream activation sequences (UAS)

• Cis-acting DNA sequence that regulates transcription

• Located upstream of promoter

• Activator proteins can bind here to enhance transcription

The GAL system in yeast

• No galactose=no gene expression. Gene expression high when galactose is present

• Structural genes for metabolism of galactose: GAL1, 2, 7, 10, etc.

• Regulatory proteins: GAL3, GAL4, GAL80

• GAL4: Generally an activator protein. If mutated, no gal gene induction occurs. Homodimer, binds 17bp region

• Promoter regions of GAL1, 2, and 7 have binding sites for GAL4

How does GAL4 induce gene expression?

• It has 2 domains: the DNA binding domain, and the activating domain

• The activating domain binds the mediator complex (coactivator), which attracts/binds RNA polymerase

GAL80

Protein that inhibits GAL4 by binding its activation domain when no galactose is present

Ho genes (in yeast)

• Gene that codes for an endonuclease responsible for switching between mating types in yeast

• Creates a break at mating type locus (MAT) and swaps in HMLα (silent MATα) or HMRa (silent MATa) copy

• Results in transcriptional gene silencing

Transcriptional gene silencing

When a gene is prevented from being transcribed, often through histone modification (ex. methylation)

Nucleosome

About 150 bp of DNA wrapped around a histone octamer (a complex made of 2*(H2B,H2A,H3,H4)) and a linker H1 that holds the DNA together

Epigenetic inheritance

Offspring inherit both DNA sequence and packing (histones)

Chromatin remodelling

Changing the position of regulatory elements and gene expression levels by moving or replacing histone octamers

Nucleosome free region (NFR)

150 bp region without nucleosomes, containing transcriptional start site

Topologically associating domains (TADs)

• Regions of DNA within a loop that interact more with each other than genes outside of the loop

• Ensures that enhancers interact with the correct promoters

• Flanked by enhancer blocking insulators

CTCF (CCCTC-binding factor)

• Proteins that bind to insulator DNA elements with CCCTC motifs

• Help maintain TADs by anchoring the chromatin loops

• Also blocks the spread of heterochromatin to euchromatic regions

Enhancer blocking insulators

• DNA sequences that bind proteins that block enhancers from activating promoters

• Can loop DNA and position enhancer away from the promoter

• Able to influence more than one gene at once

Bromodomain

• Created by acetylation

• Functional part of a protein that recognizes and binds to acetylated lysine residues in histone tails

• Increases the affinity of transcription factors for a particular gene

• Has nothing to do with bromine :(

Writer enzymes

Enzymes that add histone and DNA modifications (acetylation, methylation, phosphorylation)

Eraser enzymes

Enzymes that remove histone and DNA modifications

Chromatin modification

Altering the structure of amino acids in histones or nucleotides in DNA to impact the recruitment of transcription factors and coregulators to chromatin

Histone tails

• Chains of amino acids that stick out from the nucleosome core (the histone octamer wrapped in DNA)

• Rich in lysine and arginine (positively charged)

• Attach to phosphate group of DNA, stabilizing the DNA and bringing the nucleosomes closer together

Histone acetyltransferase (HAT)

• Transcriptional activator in yeast

• Acetylating the lysine residues of histone proteins opens the DNA up a bit, converting it from heterochromatin to euchromatin

• This allows transcription, promoting gene expression

Histone deacetylase (HDACS)

• Corepressor

• Removes acetyl groups from lysine residues on histones, converting the DNA from euchromatin to heterochromatin

• Represses transcription and therefore gene expression

Histone code

• Hypothesized by Allis and Jenuwein

• States that DNA transcription is regulated by modifications to histones, such as by HATs or HDACs

Hyperacetylated

Active genes, genes with acetyl groups to prevent formation of heterochromatin

Hypoacetylated

Inactive genes, minimal acetylation, allows the formation of heterochromatin

How does methylation impact gene expression?

• Typically decreases gene expression

• Its impact isn’t as straightforward as acetylation

• Impacts gene expression by recruiting proteins involved in gene repression or by inhibiting the binding of transcription factors

CpG islands

• Unmethylated 200bp-4kb regions of DNA

• Rich in C and G

• Near promoters

• Correlated with open chromatin (euchromatin) and active transcription

Hemimethylation

When one strand of DNA of a CpG island is unmethylated, but the complementary strand IS methylated. This can occur when the new daughter strand is the temporarily unmethylated strand

Enhanceosome

• Complex of multiple regulatory proteins that forms at enhancer regions

• Bends/alters the DNA conformation

Position effect variegation (PEV)

• Phenomenon where genes that are relocated or inverted on a chromosome can be silenced (used to be euchromatin, but in new location is close to heterochromatic centromere, which can spread and silence the gene)

• Ex. the white+ gene on Drosophila X chromosome. Causes patches of red and white in the eyes

Su var

• Suppressors of variegation, reduce variegation spread (meaning that fewer white+ genes are silenced, eye is more red)

• A trimethyltransferase (adds CH₃ to H3K9 (lysine 9 on histone 3))

E(var)/Heterochromatin protein-1 (HP-1)

• Enhances the spread of variegation by inducing heterochromatin formation, more white+ genes are silenced, eye is more white)

• A methyltransferase

Methyltransferase (HMTase)

• Enzyme that adds methyl group to lysine (K) 9 in histone 3 tail

• Forms heterochromatin, which can spread and inactivate adjacent genes

Barrier insulators

DNA sequence that is bound to by insulator-binding proteins (such as CTCF), which prevents the spread of heterochromatin

Genomic imprinting

The expression of certain autosomal genes depends on which parent they were inherited from. The repressed genes are methylated, and active genes are unmethylated

Maternal imprinting

Genomic imprinting where the mother’s copy is inactive

Paternal imprinting

Genomic imprinting where the father’s copy is inactive

Imprinting control region (ICR)

Region of DNA between Igf2 and H19 that interacts with CTCF. Is methylated in males and unmethylated in females

Barr body

An inactivated X chromosome, basically compacted as heterochromatin to prevent expression of the genes on the chromosome. This process is prevented in the presence of a Y chromosome

X inactivation

• The inactivation of one of the two X chromosomes present in females

• One X chromosome is turned into a Barr body

• Mechanism of dosage compensation in mammals

• Physically how though COME BACK TO THIS ONE

Chromosome mutation

A large-scale change in the structure or number of chromosomes, easily identifiable through karyotype/microscopy

Euploidy

The condition in which an organism has a normal number of whole sets of chromosomes for that type of organism

Monoploid

Organism that has one set of chromosomes (1n)

Diploid

Organism that has two set of chromosomes (2n)

Triploid

Organism that has three sets of chromosomes (3n)

Tetraploid

Organism that has four sets of chromosomes (4n)

Genetic load

The decreased fitness of a population due to multiple deleterious (often recessive) alleles not compensated for because they only have one copy of each gene (occurs in monoploids)

Autopolyploidy

3 or more sets of homologous chromosomes originating from the same species

What causes autopolyploidy?

An error in meiosis (non-disjunction) or mitosis

Homologous

Similar (referring to chromosomes), typically in gene content/location and size

Aneuploidy

The presence of an abnormal amount of chromosomes for the species of an organism. The first part of the word means “change”

What does colchicine do/what is it used for?

Induces polyploidy in plants by preventing the formation of microtubules during cell division, which prevents cytokinesis, essentially doubling the chromosomes in a cell

Polyploidy

Possessing more than two sets of chromosomes

Allopolyploidy

Type of polyploidy where the multiple sets of chromosomes originate from different species

Homeologous

Chromosomes that are partially similar. In allopolyploids, these chromosomes from multiple species that are not completely homologous may still pair up during metaphase (but not as well as homologous chromosomes)

Amphidiploid

• An organism possessing two full sets of chromosomes, each from a different species

• This is considered a type of allopolyploid

• They’re usually fertile

Parthenogenesis

• Form of asexual reproduction

• Unfertilized egg develops into an embryo, often when the egg becomes diploid by duplicating its chromosomes

• Common in invertebrates (likes ants) but can occur in some vertebrates, like certain lizards and fish

Trisomic

• 2n+1 copies of a chromosome

• Ex. AA BB CCC (diploid with 3 sets of chromosomes, trisomy C)

Monosomic

• 2n-1 copies of a chromosome

• Ex. AA BB C (monosomy C)

Nullisomic

• 2n-2 copies of a chromosome

• Ex. AA BB .. (nullisomy C, both copies are absent)

Nondisjunction

An error in chromosome pairing during meiosis/mitosis, occurring at the first or second division

Trisomy 21

An extra copy of chromosome 21 caused by nondisjunction

Gene balance

• Refers to whether or not an organism receives the correct number of genes

• Not directly talking about the correct number of chromosomes, but if you don’t have the right number of chromosomes you’re also not going to have the correct number of genes

Gene dosage

The number of copies of a gene present in an organism

Dosage compensation

To compensate for the unequal number of X chromosomes and therefore the potential unequal protein production, in fruit flies, the male’s X chromosome is hyperactivated, transcribing twice the amount of protein that a single X chromosome in a female does. In mammals, only one X chromosome is transcriptionally active in each cell (usually)

Klinefelter syndrome

• XXY

• Males with lanky builds, slightly impaired IQ, sterile

Why does XXY result in an abnormal phenotype?

The presence of the Y chromosome prevents the second X chromosome from being turned into a Barr body like would typically be done in XX individuals. This results in twice the normal amount of protein coded for by the X chromosomes being produced

Turner syndrome

• XO

• 1/5000 female births

• Short stature, webbed neck, decreased ovarian function, heart defects

• Typically normal X protein levels, but the missing inactivated X chromosome (Barr body) would still have a few partially transcriptionally active genes, and those missing genes account for the distinct phenotypes associated with the syndrome. The Y chromosome in males makes up for the genes still expressed on the Barr body

What must a chromosome possess to survive chromosomal rearrangements during meiosis?

One centromere and two telomeres

Acentric

• A chromosome that lacks a centromere

• Caused by certain chromosomal rearrangements

• The chromosome won’t be dragged to the poles of the cell by spindle fibers during anaphase (of mitosis or meiosis), meaning the chromosome will not be integrated into the nuclei of either daughter cell

Dicentric

• A chromosome that has 2 centromeres

• Caused by certain chromosomal rearrangements

• Each centromere will likely be pulled towards separate poles, and the chromosome will likely not be incorporated into the nuclei of either daughter cell

Anaphase bridge

Forms when the centromeres of a dicentric chromosome are pulled toward either end of the cell during anaphase. If the chromosome breaks, the pieces will each only have one telomere and the chromosome won’t be able to replicate properly

Deletion

• The loss of a chunk of a chromosome

• 2 breaks in the chromosome result in a segment of DNA being removed, and the 2 ends flanking the removed region are rejoined

Duplication

• An extra copy of a segment of a chromosome is produced

• Repeats are located either adjacent to each other or somewhere else in the genome

Inversion

• A segment of DNA is chopped out of the chromosome, flipped around, and reinserted

• Ex. abcde→adcbe