BIO103 Exam 3

Simple diffusion

Passive transport (does not require ATP)

No integral membrane protein involved

Moves molecules along their concentration gradient, from high to low concentration

Small, nonpolar, or hydrophobic molecules (e.g., O2, CO2, lipids)

Facilitated diffusion

Passive transport (does not require ATP)

Requires integral membrane transport proteins

Moves molecules down their concentration gradient, from high to low concentration

Polar or hydrophilic molecules (e.g., Na+, Ca2+, glucose)

Active transport

Requires energy input from ATP

Requires integral membrane protein pumps

Moves molecules against their concentration gradient, from low to high concentration

Polar or hydrophilic molecules (e.g., Na+, Ca2+, glucose)

Osmosis

The passive movement or diffusion of water across a semipermeable membrane

Hypertonic

Describes a solution with a higher solute concentration compared to another solution

Hypotonic

Describes a solution with a lower solute concentration compared to another solution

Endocytosis and exocytosis

Active transport mechanisms (they require ATP)

Result in particles being enclosed inside a membrane-bound intracellular sac or vesicle

Phagocytosis

Transports large particles (e.g., whole cells, debris)

High specific process (e..g, involving receptor binding and opsonization by immune cells)

Involves the extension of pseudopodia (cytoplasmic extensions) and forms large vacuoles

Does not typically involve clathrin

Pinocytosis

Transports small particles ands extracellular fluids (non-specific cell drinking)

Non-specific and does not require specific receptor binding

Does not form pseudopodia, forms small intracellular vesicles

Receptor-mediated endocytosis

Transports specific small molecules/ligands (e.g., proteins, specific viral particles) bound to membrane receptors

Specific for its target ligand and always uses receptors

Relies on clathrin coats (forming clathrin-coated pits and vesicles) and proteins like dynamin to pinch off the vesicle

The cytoskeleton & actin dynamics

Actin filaments (microfilaments) assemble and disassemble, requiring ATP

Drugs like Latrunculin A prevent the recognition of actin filaments, which disrupts cellular processes like viral entry

Role of myosin

Acts as the motor protein or “train” that walks along actin filament “tracks” (using ATP and calcium) to transport cargo such as endosomes and viral particles inside the cell

Knocking out myosin heavy chain genes (such as MYH9) heavily decreases viral entry, proving that myosin is required

Case study: SARS-CoV-2 entry

SARS-CoV-2 infects host cells by binding its Spike protein (specifically the S1 subunit and receptor binding domain) to the host cell receptor ACE2

The virus enters the host via receptor-mediated endocytosis, utilizing clarthin-coated pits

Inhibiting clathrin pit formation (using drugs like Dynasore or Pitstop 2) significantly reduces spike protein and viral uptake

Transcription

Process where RNA polymerase synthesizes RNA (including pre-mRNA) using a DNA template

Template strand

The DNA strand read by RNA polymerase in a 3’ to 5’ direction during transcription

Coding (non-template) strand

The DNA strand complimentary to the template strand, possessing the same sequence as the resulting pre-mRNA (except with T instead of U)

Promotor

A double-stranded DNA region (cis-element) located at the 5’ end of the coding strand where RNA polymerase binds to initiate transcription

Terminator

The DNA region at the 3’ end of the coding strand where transcription stops and RNA polymerase detaches

Transcription factors

Regulatory proteins (trans-acting factors) that bind to promoters, enhancers, or silencers to promote or inhibit transcription

Pre-mRNA modifications

In eukaryotes, primary RNA transcripts undergo essential processing steps in the nucleus before translation

5’ cap

A modified guanine nucleotide added to the 5’ end of the pre-mRNA that helps regulate nuclear transport, protects the mRNA from degradation, and promotes translation

Poly(A) tail

A string of adenine nucleotides added to the 3’ end of that aids in nuclear transport, prevents degradation, and assists in translation

Exons

Expressed coding regions of nucleic acid that are retained and spliced together during RNA processing

Introns

Intervening, non-coding regions of nucleic acid that are spliced out of pre-mRNA prior to translation

Translation

The process where ribosomes synthesize a polypeptide chain using mature mRNA as a template

Involves ribosomes, mRNA, amino acids, tRNAs, and aminoacyl-tRNA synthetase (enzymes responsible for attaching amino acids to their corresponding tRNAs)

Codon

A sequence of three mRNA nucleotides (read 5’ to 3’) that specifies a particular amino acid or serves as a stop signal

Transfer RNA (tRNA)

An RNA adapter molecule featuring an anticodon that matches mRNA codons and delivers the correct amino acid to the growing chain

Reading frame

The specific triplet grouping of codons read by the ribosome during translation, bounded by a start codon (AUG/ATG) and a stop codon (UAA/TAA, UAG/TAG, UGA/TGA)

Three steps of translation

1. Initiation: Assembly of the ribosome, the first charged tRNA, and the mRNA strand

2. Elongation: Sequential addition of amino acids as tRNAs deliver them to the ribosome

3. Terminator: A stop codon is reached, recruiting a release factor that causes the translation complex to dissociate

Ribosome anatomy

Composed of a small ribosomal subunit and a large ribosomal subunit, containing three primary tRNA binding sites: A site, P site, and E site

Polypeptide polarity

Polypeptides are synthesized from the N-terminus (amino terminal end) to the C-terminus (carboxyl terminal end), corresponding to the 5’ to 3’ direction of the mRNA and non-template DNA strands

Open reading frames (ORFs)

A continuous stretch of codons starting with a 5’ AUG start codon and terminating with a stop codon (3’ UAA, UAG, or UGA) in the same reading frame

The one with the longest polypeptides is typically the functional, correct one

Bacteria

Single-celled organisms with ribosomes and a nucleoid

Consist of circular double-stranded DNA

Reproduce asexually via binary fission

Infections can be treated using antibiotics (e.g., penicillin, which targets peptidoglycan cell wall synthesis)

Viruses

Non-living biological entities with zero cells and organelles

Highly diverse and can be dsDNA, ssDNA, dsRNA, or ssRNA

Infects host cells and hijacks host machinery to replicate

Treated by targeted antivirals, such as nucleoside analogs (e..g, AZT for HIV), which mimic natural nucleosides and prematurely terminate viral DNA replication via reverse transcriptase inhibition

SARS-CoV-2 life cycle

1. The viral Spike protein binds to the host cell receptor ACE2

2. Viral entry occurs via direct membrane fusion or receptor-mediated endocytosis

3. The viral RNA genome is released into the host cell cytoplasm

4. Viral RNA is translated into large polyproteins using host ribosomes

5. Viral proteases perform proteolysis, cleaving polyproteins into functional viral proteins

6. Viral RNA-dependent RNA polymerase (RdRp) replicates the viral genome

7. New viral components undergo packing and assembly

8. Progeny virions are transported to the plasma membrane and released from the cell

SARS-CoV-2 genomic features

A positive-sense single-stranded RNA (+)ssRNA) virus that structurally mimics eukaryotic mRNA by featuring a 5’ cap, a 3’ poly(A) tail, and a Kozak consensus sequence

Polyprotein processing

Because the viral genome encodes multiple proteins from a single transcript despite having few Kozak sequences, it relies on specific amino acid cleavage sites recognized by viral proteases to generate individual structural and non-structural proteins

Positive-sense ssRNA ((+)ssRNA)

A single-stranded RNA genome that mimics cellular mRNA

Because of this similarity, viral genes can be directly translated by host cell ribosomes once inside the cell

It shares structural features with eukaryotic mRNA including a 5’ cap, a 3’ poly(A) tail, and Kozak consensus sequences

Negative-sense ssRNA ((-)ssRNA)

A viral single-stranded RNA genome whose sequence is complimentary (the opposite strand) to viral mRNA

Unlike positive-sense RNA, negative-sense RNA cannot be translated directly by host cell ribosomes

The virus must carry an RNA-dependent RNA polymerase enzyme inside its particle to transcribe the (-)ssRNA into a readable positive-sense((+)ssRNA) template before any viral proteins can be synthesized

Kozak sequences

Nucleic acid motif that acts as a “location” signal, telling the ribosome where to start translation

One of the key elements required for translation to occur in eukaryotic cells

Viruses utilize Kozak sequences within their RNA genomes to direct host ribosomes to initiate the translation of viral proteins

Hallmarks of inflammation

Redness (rubor): Caused by the dilation of blood vessels

Swelling (tumor): Caused by fluid and cells migrating into the tissue

Pain (dolor): Caused by swelling and chemical mediators interacting with sensory receptors

Heat (calor): Caused by chemical mediators elevating body temperature

Pathogen-associated molecular patterns (PAMPs/MAMPs)

Macromolecule specific to pathogens (such as bacteria and viruses) that are absent in the host

Pattern recognition reactors (PRRs)

Receptors that recognize specific PAMPs/MAMPs

Plasma-membrane-associated TLRs

Detect extracellular pathogens (e.g., TLR-4 detects lipopolysaccharide or LPS from Gram-negative bacteria)

Endosome-membrane-associated TLRs

Detect intracellular pathogens and viral components (e..g, TLR-3 for dsRNA, TLR-7/8 for ssRNA and TLR-9 for CpG DNA)

G Protein-coupled receptors (GPCRs)

7-transmembrane receptors that respond to water-soluble signaling molecules (such as cytokines and hormones)

IL-8 signaling in neutrophils

1. The cytokine IL-8 binds to the CXCR1 receptors (a GPCR)

2. The receptor activates associated G proteins (such as G alpha q)

3. Activated G proteins stimulate enzymes such as phospholipase C or adenylyl cyclase

4. Produces second messengers (e,g., cAMP or IP3, DAG, and Ca2+) that amplify the signal

5. Activates downstream kinases (such as PKA or PKC) leading to cellular responses like actin-myosin shape changes and chemotaxis

Ligand-gated ion channels

Allow ions influx (facilitated diffusion) to serve as a signal or trigger phosphorylation cascades

Water-soluble receptor

Receptor tyrosine kinases (RTKs)

Dimerize and autophosphorylate tyrosine residues upon ligand binding to initiate signal-transduction cascades

Water-soluble receptor

Kinases

Enzymes that add phosphate (PO4) groups (typically to serine, threonine, or tyrosine residues), frequently activating target proteins

Phosphotases

Enzymes that remove phosphate groups from proteins, typically turning off phosphorylation cascades and de-activating pathways

ERK1/2 MAPK pathway

Stimuli: Growth factors, mitogens, GPCR agonists

Cascade: Raf (MAPKKK) to MEK (MAPKK) to ERK (MAPK)

Cellular response: Cell growth, survival, differentiation, and development

p38 MAPK pathway

Stimuli: Cellular stress, inflammatory cytokines, or LPS signaling through TLR-4

Cascade: TAK1 (MAPKKK) to MKK3/5 (MAPKK) to p38 (MAPK)

Cellular response: Inflammation, apoptosis, growth, and differentiation

Lipid-soluble hormones

Receptor location: Intracellular (cytosol or nucleus)

No second messengers

No phosphorylation cascade

The hormone-receptor complex acts directly as a transcription factor to alter gene transcription

E.g., dexamethasone, steroids

Water-soluble molecules

Receptor location: Plasma membrane or endosomal membrane

Second messengers produced: cAMP. IP3, DAG, Ca2+

Uses extensive phosphorylation cascade like MAPK pathways

Triggers downstream transcription factors to modify gene expression

E.g., LPS, cytokines

SARS-CoV-2 spike protein

The SARS-CoV-2 spike protein S1 binds to host receptors (like ACE2) and stimulates monocytes/macrophages to produce a “cytokine storm” (excessive release of pro-inflammatory cytokines such as IL-9, TNF, and IL-1 beta)

Mechanism of dexamethasone

It is a synthetic glucocorticoid (steroid hormone) used to treat severe COVID-19 inflammation

It inhibits the p38 MAPK pathway (evidenced by reduced p38 phosphorylation) and dampens pro-inflammatory cytokine production

It reduces inflammation like a “leaky faucet” rather than completely shutting off the immune system, allowing the body to maintain a controlled immune response against the infection

mRNA isolation

Total RNA or mRNA is isolated from cells, often utilizing an oligo-dT column containing thymine stretches that bind to the 3’ poly(A) tail of mature eukaryotic mRNA

cDNA synthesis

Reverse transcriptase is used to synthesize complimentary DNA (cDNA) using the mRNA template

Conventional PCR

DNA polymerase then amplifies specific target sequences from the cDNA template

Qualitative vs. quantitative

Standard RT-PCR is qualitative or semi-quantitative but it can be quantitative (qRT-PCR) by incorporating fluorescent probes to monitor amplification in real time

Quantitative RT-PCR (qRT-PCR)

Real time detection: Fluorescence is measured as PCR product accumulates during the exponential phase when reagents are in excess

Threshold (Ct)

The threshold is a baseline set to intersect the exponential phase of amplification

Ct (threshold cycle) is the exact cycle number where the fluorescence signal crosses the threshold

A lower Ct value means the signal crossed the threshold earlier, which indicates a greater initial amount of target mRNA in the sample

Antibody structure

Composed of heavy chains and light chains linked by disulfide bonds

Variable region/antigen-binding site: Specifically recognizes and binds to target epitopes

Fc region: Mediates interactions with immune cells and receptors

Reducing agents in SDS-PAGE

Treating an antibody with a reducing agent disrupts the disulfide bonds, allowing the individual polypeptide chains (heavy and light chains) to separate and appear as distinct bands on an SDS-PAGE gel

Direct ELSA

Detects the presence of antigens (such as viral proteins)

Uses capture antibodies and enzyme-labeled detection antibodies that recognize different epitopes on the target antigen

Indirect ELSA

Detects the presence of antibodies produced by the patient against a pathogen or vaccine

Why are microtiter plate ELSA quantitative?

They include a standard curve generated from serially diluted purified target molecules

Lateral-flow ELSA (at-home tests)

Qualitative tests using capillary flow on a strip to give rapid results via test lines (positive) and control lines (valid test)

Gene targets in SARS-CoV-2 assay

N1 & N2: Specific SARS-CoV-2 viral genes

N3: Shared gene from related coronaviruses (e.g., SARS-CoV)

RP (human control): A human gene confirming successful sample collection and RNA extraction

Control sample purposes

Human control: Positive control for human mRNA isolation/amplification; negative control for viral primers

Viral genome control: Positive control confirming reverse transcriptase and PCR reagents work for the virus

Water control: Negative control to check for reagent contamination

Community monitoring

Sewage analysis involves sampling, sample concentration, RNA extraction, and RT-qPCR to track community-level viral prevalence and public health trends