Lecture Notes on Genetics

Flashcards on gene structure, RNA processing, DNA replication, DNA binding proteins, Lac operon, Tryptophan biosynthesis, Catabolite Repression, Myc/Max System, Galactose System, and Post-Translational Modifications.

Promoter

DNA sequence where RNA polymerase and transcription factors bind to initiate transcription; the 'on switch' of a gene.

Intron

Non-coding sequence in a gene, transcribed into pre-mRNA but removed during RNA splicing.

Exon

Coding sequence in a gene, expressed and make proteins, transcribed into pre-mRNA and kept during RNA splicing to form mature mRNA

5' capping

7-methylguanosine cap is added to the 5' end of mRNA to protect RNA and help export of genetic material from the nucleus

3' polyadenylation

Poly-A-tail (\~100-250 adenines) is added to the 3' end to increase stability and enhance translation for final amino acid chain

RNA splicing

Introns are removed and exons are joined together by the spliceosome to produce a continuous coding sequence for protein synthesis

Alternative splicing

Different combinations of exons are combined to form multiple mRNA isoforms, increasing protein diversity from a single gene

Nuclear export

Mature mRNA is transported to cytoplasm, causing mRNA to be translated into protein (final products)

RNA Editing

Nucleotides in RNA sequence are chemically modified, allowing protein coding to be altered for specific genes

rRNA

Structural part of ribosomes

mRNA

Carries protein-coding information of strand

tRNA

Transfers amino acids

snRNA

Splicing - removal of introns

Initiation (DNA Replication in Prokaryotes)

RNA polymerase binds to promoter with transcription factors causing DNA to unwind

Elongation (DNA Replication in Prokaryotes)

RNA polymerase synthesizes RNA in direction of 5' - 3'

Rho-independent Termination

Rho protein is not required, instead relying on a GC hairpin loop in the RNA sequence and a string of US

1. RNA Polymerase transcribes a region that forms a GC-rich hairpin loop in RNA

2. Loop causes polymerase to pause

3. Weak U-A bands found in RNA and DNA forms are unstable

4. RNA dissociates and transcription ends

Rho-dependent Termination

Rho protein (helicase) required, no hairpin needed

Helix-turn-helix

Structure consists of two α-helices that are connected by a short turn with one helix fitting in the DNA groove. Function is to regulate gene expression and is found during DNA repair, RNA metabolism, and protein interactions

Zinc Finger

Structure has one or more zinc ions that stabilize the DNA fold by binding to DNA in the major groove.

Leucine Zipper

Structure has leucine after every 7 amino acids to form a zipper that enables the binding of protein subunits to DNA

Lactose

Disaccharide sugar that is primary energy source of E. coli

β-galactosidase (LacZ)

Enzyme responsible for hydrolyzing lactose into glucose and galactose

Permease (LacY)

Membrane protein that transports lactose into the cell

Operator (O)

DNA sequence where the repressor will bind

Transacetylase (LacA)

Enzyme that transfers an acetyl group from acetyl-CoA to galactose to form galactosidate

LacI⁺

Normal repressor that binds to the operator and blocks transcription from occurring

LacIˢ

Superrepressor, cannot bind allolactose so will always bind to the operator, keeping lac genes OFF

LacOᶜ

Constitutive operator, repressor can't bind, lac genes always ON (LacO⁻)

LacIᵈ

Dominant negative repressor, interferes with normal LacI⁺; even in the presence of functional LacI⁺ genes, it can interfere with the normal repressor's ability to bind to operator, causing constitutive expression of genes

LacIᵗᵇ

Temperature-sensitive LacI, functional at one temperature and inactive at another

LacI⁻

Defective repressor that cannot bind to operator

z⁺

Functional β-galactosidase gene

z⁻

Non-functional β-galactosidase gene

O⁺

Normal operator where repressor will bind

Tryptophan Biosynthesis Attenuation

Mechanism for reducing the expression of the trp operon when levels of tryptophan are high. If tryptophan is present, transcription stops early. Relies on a leader sequence with 4 regions that form stem-loop structures. High Trp: ribosome doesn’t pause which forms 3-4 terminator loopsand transcription stops. Low Trp: ribosome stalls at Trp codon forming 2-3 antiterminator loops and transcription continues.

cAMP

Signaling molecule that binds to CAP

Adenylate cyclase

Enzyme responsible for converting ATP to cAMP

Catabolite Repression

Mechanism where the presence of a preferred carbon source inhibits the expression of genes involved in using other carbon sources; in the case of the lac operon in E. coli, it ensures bacteria metabolize glucose before utilizing lactose

Enhancers

DNA sequences located far from the promoter that increase transcription efficiency; bound by activator proteins

TATA Box

Core promoter sequence (TATAAA) located 25-30 bp upstream from transcription start site; binds TATA-binding protein (TBP) to help form the basal transcription complex

Basal Transcription Machinery

RNA Polymerase II and general transcription factors needed for transcription from a promoter

Blocking

Repressor binds directly to the activator's binding site on DNA preventing activator binding

Quenching I

Repressor binds directly to the activator protein's DNA-binding domain, preventing it from binding DNA

Quenching II

Repressor binds to the activator's activation domain, preventing recruitment of transcriptional machinery

Myc

A transcription factor within the activation domain, forms a heterodimer with Max to activate the transcription of growth-promoting genes

Coactivators

Proteins that don't directly bind to DNA but bridge transcription factors to RNA Pol, aiding in chromatin remodeling and recruiting RNA Pol II

Homodimer

Two identical subunits

Heterodimer

Two different subunits

Heteromer

Multi-subunit complex with diverse components

Gal4

A transcription activator that binds to the upstream activating sequence (UAS) to promote GAL gene transcription

Ubiquitination

Ubiquitin attaches to a protein causing it to be tagged for degradation by the proteasome; the protein is degraded and recycled

Phosphorylation

Addition of a phosphate group to amino acids; used in DNA repair, gene expression, and cell signaling

Activator

Transcription factor, binds to DNA and recruits chromatin remodeling complexes to open chromatin structure and enhance transcription

Silencers

DNA sequence where repressors bind

Acetylation

Histone acetyltransferases (HATs) add acetyl groups to histones; this modification typically leads to a more open and accessible chromatin structure, facilitating transcription

Deacetylation

Histone deacetylases (HDACs) remove acetyl groups from histones; this modification generally leads to a more condensed chromatin structure, reducing transcription

DNA Methylation

DNA methyltransferases (DNMTs) add methyl groups to DNA, typically at cytosine bases; this modification is often associated with transcriptional repression

Demethylation

Demethylases remove methyl groups from DNA, reversing the effects of DNA methylation and potentially leading to increased gene expression

Methylation

Addition of methyl groups to histones; can activate or repress transcription

Kinases

Enzymes that phosphorylate proteins, altering their activity

Phosphatases

Enzymes that remove phosphate groups from proteins, reversing the effects of phosphorylation

miRNA

Small non-coding RNA molecules involved in post-transcriptional regulation of gene expression

Antisense RNA

RNA molecules transcribed from the antisense strand of a gene, can inhibit gene expression by binding to mRNA

RNA-binding proteins (RBPs)

Proteins that bind to mRNA and affect its translation, stability, or localization

Transcription Initiation

Initiation of transcription factors and RNA polymerase to promoter, forming initiation complex. Then unwinding of DNA double helix.

Transcription Elongation

RNA polymerase moves along the template strand, synthesizing mRNA in the 5' to 3' direction.

Transcription Termination

Transcription stops when RNA polymerase reaches a termination signal (e.g., hairpin loop, Rho factor).

Translation Elongation

Ribosome binds to mRNA, tRNA brings the corresponding amino acid, forming a peptide bond between them.

Translation Termination

Ribosome reaches a stop codon on mRNA, translation stops, polypeptide chain is released.

Ubiquitination Process

addition of ubiquitin to target proteins, marking them for degradation. This is followed by recognition and unfolding of the targeted protein

Degradation by the Proteasome

the proteasome degrades the tagged protein into smaller peptides and amino acids, which are then recycled.

Kinases action on proteins

Kinases add phosphate groups to proteins, altering their activity and function, either activating or inactivating them.

Protein-mediated cell signaling

Phosphorylated proteins participate in cell signaling pathways, mediating cellular responses.

Effect of Activation of Gene Expression

Demethylation can lead to increased accessibility of DNA and activation of gene expression.

Antisense RNA

Regulatory RNA that is complementary to an mRNA sequence. Binds to the mRNA, blocks ribosome binding or causes degradation, and inhibits translation. Used by plasmids and phages for post-transcriptional regulation.

Feedback Inhibition

end product of a biosynthetic pathway inhibits the first enzyme of the pathway, preventing waste of energy/resources when sufficient product is present.

Enhancers

DNA sequences located far from the promoter that increase transcription efficiency; bound by activator proteins that help recruit transcription machinery to the promoter.

TATA Box

A core promoter sequence (TATAAA) located 25-30 bp upstream from the transcription start site; binds the TATA-binding protein (TBP) to help form the basal transcription complex.

Introns

Non-coding sequences removed during RNA processing.

Exons

Coding sequences that are spliced together to form mature mRNA.

Basal Transcription Machinery

Includes RNA polymerase II and general transcription factors that are required for minimal transcription from a promoter.

Quenching I

A repressor binds directly to the activator protein’s DNA-binding domain, preventing it from binding DNA.

Quenching II

A repressor binds to the activator’s activation domain, preventing recruitment of the transcriptional machinery.

Myc/Max System

Transcription factor with an activation domain; forms a heterodimer with Max to activate the transcription of growth-promoting genes; Max/Max homodimers are inactive or repressive; balance of Myc/Max and Max/Max affects cell proliferation.

Coactivators

Proteins that don’t bind DNA directly but bridge transcription factors to RNA polymerase, aiding in remodeling chromatin or recruiting RNA Polymerase II. Homodimer: two identical subunits (Max/Max). Heterodimer: two different subunits (Myc/Max). Heteromer: multi-subunit complex with diverse components (Mediator complex).

Gal4/UAS System

Transcription activator that binds to upstream activating sequence (UAS) to promote GAL gene transcription. In the absence of galactose: Gal80 binds to Gal4 and blocks activation → transcription doesn’t occur. In the presence of galactose: Gal3 binds galactose → binds Gal80 → releases Gal4 → transcription continues.

DNAse I Hypersensitivity

Regions of DNA that are active become more sensitive to DNAse I digestion.

Chromatin Remodeling

ATP-dependent complexes reposition nucleosomes, which opens chromatin structure for transcription factors to bind DNA.

DNA Methylation

Addition of a methyl group to cytosine, associated with gene silencing.

Insulator Proteins

Define boundaries of gene expression domains and block the action of enhancers on unintended promoters if placed between them.

Super Enhancers

Clusters of enhancers with high activity, drive expression of genes critical for cell identity.

RNA Interference (RNAi)

Post-transcriptional gene silencing mechanism; occurs as a dicer processes double-stranded RNA in siRNA or miRNA and loads it into RNA-induced silencing complex (RISC). Dicer cuts long double-stranded RNA into shorter fragments. RISC uses the RNA guide to bind complementary mRNA and causes mRNA cleavage and translation inhibition or degradation.

Ubiquitination

Attachment of ubiquitin to a protein, tagging it for degradation by the proteasome; important for regulation of protein levels and for the removal of misfolded proteins.

Phosphorylation

Addition of a phosphate group to amino acids, regulates protein activity, localization, or interactions that are common in signalling cascades.

Levels of Regulation

Bacteria have mostly transcription initiation, while eukaryotes have multiple levels (chromatin remodeling, transcription, RNA processing, translation, post-translational modifications).

Gene Expression Cascades

Basal expression in bacteria occurs when the promoter is weakly bound by RNA polymerase without regulation. Basal expression in eukaryotes requires general transcription factors & RNA pol II for basal expression. Activated expression in bacteria have enhancers or activators that increase transcription by helping RNA polymerase bind. Activated expression in eukaryotes cause activators to bind enhancers and recruit co-activators and chromatin modifiers.

DNA Binding Proteins

Bacteria mainly use helix-turn-helix to bind operators near the promoters. Regulators include repressors and activators. Eukaryotes have a wide variety of proteins they utilize to bind enhancers or silencers. Regulators include transcription factors, co-activators and repressors.

Operons & Gene Organization

Bacteria contain their genes within operons (lac operon, trp operon) and have multiple genes under one promoter. Eukaryotes do not contain operons and each gene contains its own promoter.

Initiation of Transcription

In bacteria, the binding of RNA polymerase to the promoter initiates transcription, and RNA polymerase is recruited by σ factor to make it bind directly. In eukaryotes, initiation of transcription occurs through the TATA box which is recognized by specific transcription factors, and RNA polymerase is recruited by multiple general transcription factors and mediator complex.

Transcription & Translation

Bacteria have coupled processes because translation starts while mRNA is being made. No processing is necessary, and mRNA is used as is. Eukaryotes have separate processes because the mRNA has to be exported from the nucleus to the cytoplasm. Requires extensive processing that includes the 5’ cap, poly(A) tail, and splicing.