Module 2 Notes: Molecular Genetics and Disease
Genome organization and central dogma
Core components of molecular genetics covered: genome organization, transcription, RNA splicing/processing, turnover and quality control, translation, RNA modification, and RNP assembly.
Key schematic cues:
GCCAUG motif indicating initiation context near start codon (AUG).
NH₂ represents the amino terminus of a polypeptide.
Processes in order of flow from DNA to functional products: transcription → RNA processing → translation → protein folding/assembly → post-translational modifications and turnover.
Subcellular focus: RNA metabolism and ribonucleoprotein (RNP) complexes drive maturation and quality control of transcripts.
Protein basics: structure, function, and examples
Proteins are central to all living processes and perform diverse roles:
Enzymes: biological catalysts that accelerate cellular reactions.
Structural components: scaffolding for membranes, filaments, bone, hair.
Transport: carriers for substances.
Regulatory, signaling, and defense: regulators and antibodies.
Protein primary structure determines higher-level organization:
Primary structure: amino acid sequence.
Secondary/tertiary structures arise from the primary sequence.
Quaternary structure forms when multiple polypeptides assemble (e.g., hemoglobin).
Peptide bond formation:
Each amino acid has a central carbon linked to an amino group (–NH₃⁺), a carboxyl group (–COO⁻), a hydrogen, and a side chain (R).
Peptide bonds covalently join amino acids via condensation between the carboxyl of one and the amino group of the next.
Notable example proteins:
Silk fibroin: uses three smallest amino acids to form beta sheets, conferring very high tensile strength.
GFP: a bioluminescent protein from jellyfish used as a fluorescent marker.
Firefly luciferase: catalyzes oxidation of luciferin in the presence of O₂ and ATP to emit light via a luciferin-adenylate intermediate.
Protein structure in action: folding and organization
Protein folding is organized across levels:
Secondary and tertiary structures are determined by the primary sequence.
Quaternary structure arises when two or more polypeptides assemble (e.g., hemoglobin).
Membrane proteins and cellular localization
Membrane proteins are critical for communication and transport:
Integral membrane proteins: embedded within the lipid bilayer; may be linked to lipids or fatty acids.
Peripheral membrane proteins: associated with the membrane surface rather than embedded.
Major roles of membrane proteins:
Recognition and binding (receptors) for substances entering or leaving the cell.
Pores or transport channels for ions, and carriers for amino acids and monosaccharides.
Enzymes that drive active pumps to regulate ion concentrations (e.g., maintaining high intracellular K⁺ while keeping Na⁺ low outside).
Cell surface markers (glycoproteins) for cell–cell identification.
Cell adhesion molecules (CAMs) that connect cells to the cytoskeleton and to each other.
Post-translational modifications (PTMs): expanding protein diversity
Proteins undergo multiple PTMs that regulate activity, localization, interactions, and stability:
Phosphorylation: can activate or inactivate proteins; addition/removal by kinases/phosphatases.
Kinase: enzyme that phosphorylates proteins.
Phosphatase: enzyme that dephosphorylates proteins.
Disulfide bonds: formed and rearranged by Protein Disulfide Isomerase (PDI) to stabilize folding; rearrangements enable alternative bonding patterns.
Lipidation and glycosylation: direct proteins to membranes or specific organelles and modulate interactions.
Ubiquitination: marks proteins for degradation by the proteasome; acetylation can modulate binding sites.
PTMs contribute to protein diversity beyond the genome, enabling nuanced control of cellular processes.
References for PTMs and their strategies: Creative Proteomics blog; Nature Scitable on protein function.
Signaling pathways and cancer biology: IGF-1 and RB
IGF-1 signaling pathway:
IGF-1 ligand binds to its receptor IGF-1R at the cell membrane.
Receptor activation triggers intracellular signaling, leading to translation of new proteins that act as intracellular communicators.
Phosphorylation activates the signaling cascade; in some contexts, signaling promotes cell proliferation.
Relevance to cancer: reduced signaling at IGF-1R and downstream components is a pharmacologic strategy under investigation for cancer treatment.
IGF-1 role in cancer: promotes cell proliferation and inhibits apoptosis, correlating with higher cancer risk in epidemiological studies for breast, colorectal, and prostate cancers; serum IGF-1 can serve as a diagnostic risk marker.
Retinoblastoma protein (RB) and cell cycle control:
RB is a key tumor suppressor that blocks the G1→S transition.
In the absence of growth factors, RB is hypophosphorylated and binds gene regulatory proteins, repressing proliferation-related transcription.
Growth factors activate signaling pathways that lead to RB phosphorylation, releasing gene regulators and enabling cell proliferation.
RB mutation or loss of expression disrupts growth control, causing unregulated cell division, DNA replication errors, and genomic instability, which can lead to retinoblastoma.
PTMs in neurodegenerative diseases: phosphorylation examples
Post-translational modifications influence neurodegeneration via protein aggregation and misfolding:
Alzheimer's disease: increased phosphorylation of tau affects its aggregation and stability, forming neurofibrillary tangles and neuronal death.
Parkinson's disease: hyperphosphorylation of α-synuclein may promote Lewy body formation.
Huntington's disease: phosphorylation of mutant huntingtin (mHTT) can reduce aggregation and provide some neuronal protection.
Amyotrophic lateral sclerosis (ALS): phosphorylation of TDP-43 impacts its mislocalization and aggregation, contributing to neuronal damage.
TDP-43: a DNA/RNA-binding protein essential for gene regulation and RNA processing; pathological phosphorylation and mislocalization contribute to cytoplasmic inclusions in ALS.
DNA, RNA, and the genetic code: fundamentals
DNA and RNA overview:
DNA and RNA structures rely on phosphate diesters that render the backbone negatively charged and polar.
RNA contains a 2'-hydroxyl group, increasing polarity and solubility, and contributing to distinctive behavior in biochemical contexts (e.g., extraction). In acidic environments, RNA is more soluble, aiding separation from DNA.
Both nucleic acids carry phosphate backbones and are negatively charged; their polarity drives their aqueous solubility.
Nucleotides and bases:
DNA components: five-carbon sugar deoxyribose, phosphate, and bases: cytosine (C), thymine (T), adenine (A), guanine (G).
Pyrimidines: cytosine (C) and thymine (T).
Purines: adenine (A) and guanine (G).
The genetic code and translation fundamentals:
A codon consists of 3 nucleotides; there are 64 codons in total: 64 codons.
Of these, 61 codons encode the 20 standard amino acids.
The remaining codons include stop codons: UAA, UAG, UGA.
The start codon is AUG (codes for Methionine) and also marks the initiation of translation.
tRNAs carry specific amino acids and contain anticodons that pair with codons on mRNA during translation.
Genes, transcription, and RNA types:
Genes store information for producing all cellular proteins and RNA molecules.
Core RNA types include mRNA, tRNA, and rRNA that work together for translation.
Transcription: how RNA is made from DNA
Eukaryotic transcription is driven by RNA polymerase II and requires a multi-subunit complex with regulatory factors.
Core transcription machinery:
RNA polymerase II binds promoter regions with General Transcription Factors (GTFs) to form a Pre-Initiation Complex (PIC).
Core promoter elements include:
TATA element with consensus sequence TATAAA, recognized by TATA-binding protein (TBP) within TFIID.
BRE (TFIIB Recognition Element).
Transcription direction and strand selection:
Transcription proceeds in the 5' to 3' direction on the template strand, which is read by RNA polymerase II to synthesize a complementary RNA.
The RNA transcript has the same polarity as the non-template (coding) strand, except RNA contains uracil (U) instead of thymine (T).
Transcription unit organization:
Promoter: DNA sequence recognized by transcription machinery; defines which DNA strand serves as template and the transcription start site.
RNA-coding region: sequence copied into RNA.
Terminator: signals transcription termination.
Promoter is not transcribed.
Transcription initiation and promoter architecture:
PIC formation is driven by GTFs and promoter elements; promoter orientation determines transcription direction.
RNA processing: maturation of the primary transcript
Primary transcripts require processing in eukaryotes:
5' capping: addition of a 7-methylguanine cap at the 5' end (often denoted as 7-methylguanine).
3' poly-A tail: addition of 50–250 adenine nucleotides to the 3' end.
3' cleavage: processing truncates the 3' end prior to polyadenylation.
Splicing: introns are removed; exons are joined to form the mature mRNA.
Mature mRNA regions:
5' untranslated region (5' UTR).
Protein-coding region.
3' untranslated region (3' UTR).
Splicing implications:
Alternate (alternative) splicing generates different mature mRNA transcripts from a single pre-mRNA, expanding proteomic diversity.
From DNA to protein: translation overview
Translation machinery and components:
Ribosome: large and small subunits, mRNA, and charged tRNA carrying amino acids.
The genetic code uses 64 codons to encode 20 amino acids; tRNA anticodons pair with codons on mRNA to incorporate amino acids into a growing polypeptide.
Translation in the genetic code context:
Proteins are made from 20 amino acids.
The tRNA anticodon–codon pairing ensures the correct amino acid is added at each step.
Translation involves initiation, elongation, and termination phases.
Initiation codon and start of protein synthesis:
The initiation codon is AUG, encoding Methionine.
The ribosome-catalyzed peptide bond formation links amino acids in the order specified by the mRNA sequence.
Translation in detail: steps and machinery
Key steps of translation:
tRNA charging: aminoacyl-tRNA synthetases load specific amino acids onto their matching tRNAs (codon-specific delivery).
Initiation: ribosomal subunits assemble at the start codon (AUG) with initiator tRNA (Met) and initiation factors.
Elongation: successive amino acids are added as codons are read; peptide bond formation occurs in the ribosome.
Termination: stop codons are encountered and the polypeptide is released.
Outcome: polypeptide chain that will fold into a functional protein.
Protein folding and the endoplasmic reticulum (ER)
ER-associated processing and quality control:
The ER lumen supports four major protein-processing roles:
1) Folding/refolding of polypeptides.
2) Glycosylation of proteins.
3) Assembly of multi-subunit protein complexes.
4) Packaging of proteins into vesicles for trafficking.
Disulfide bond formation and folding catalysts:
Protein Disulfide Isomerase (PDI) rearranges disulfide bonds by transiently using cysteine residues to guide correct bonding patterns.
Correct folding may require shifting initial disulfide patterns to more stable configurations as proteins mature.
Quality control and trafficking:
Properly folded proteins exit the ER to the Golgi and then to the extracellular environment.
Misfolded proteins are detected by the quality-control system and directed to degradation via the unfolded protein response (UPR); they are ubiquitinated and degraded by proteasomes in the cytoplasm.
Molecular chaperones (including heat shock proteins, Hsp) assist in folding, assembly, and translocation processes.
Cellular adaptations, injury, and disease concepts
Definitions and concepts:
Disease: disturbance of structure or function of tissues or organs.
All diseases begin with alterations in cells.
Homeostasis: a state of stable physiological conditions; adaptation is a reversible structural/functional response to normal, stressful, or adverse conditions.
Disease-related dysfunction can be accompanied by characteristic structural changes (lesions).
Stages of cellular adaptation and injury:
Adaptation aims to maintain homeostasis; reversible if the stimulus is mild/transient.
If the stimulus persists or is severe, injury may become irreversible, potentially leading to cell death.
Major cellular adaptations:
Atrophy: decrease in cell size.
Hypertrophy: increase in cell size.
Hyperplasia: increase in cell number.
Metaplasia: reversible replacement of one mature cell type by another less mature type (can be a cancer precursor).
Dysplasia: abnormal cellular growth; not a true adaptation but an atypical hyperplasia.
Examples of hypertrophy and organ-level adaptation:
Cardiac hypertrophy involves increased synthesis and accumulation of proteins within cellular components (plasma membrane, ER, myofilaments, mitochondria).
Hypertrophy can be physiologic (e.g., skeletal muscles with heavy work) or pathologic (e.g., response to disease conditions); workload changes influence the persistence of hypertrophy.
Renal hypertrophy following nephrectomy occurs as the remaining kidney enlarges with increased workload.
Therapeutic antibodies and infectious disease control
Neutralizing antibodies as therapeutic tools:
Block ligand–receptor interactions to prevent disease-causing molecules or pathogens from binding to cellular targets.
Mechanisms include: occupying binding sites on pathogens or receptors, or binding soluble ligands to prevent receptor engagement.
Other applications include autoimmune therapies targeting cytokines like TNF-α and antiviral strategies that block viral entry.
Figures illustrating neutralization strategies (summary of mechanisms):
a) Prevent virion attachment via spike protein conformational changes.
b) Promote virion aggregation to impede attachment.
c) Directly block spike protein binding to host receptors.
d) Block membrane fusion of virus and host.
e) Block conformational changes in spike protein required for entry.
f) For endosomal entry viruses, block endosomal cleavage or receptor binding to enter cytoplasm.
g) Block viral egress from the cell.
Translational signaling and cancer biology: IGF-1 signaling
IGF-1 signaling details:
IGF-1 binds to IGF-1 receptor (IGF-1R) on the cell surface, activating intracellular signaling cascades.
Resulting phosphorylation events promote translation of new proteins, acting as intracellular communicators.
In cancer, IGF-1 signaling supports proliferation and survival (anti-apoptotic and pro-proliferative effects).
Therapeutic implications:
Targeting IGF-1 signaling and downstream pathways is a strategy for cancer therapy.
Clinical/epidemiological notes:
Serum IGF-1 levels have been positively associated with risks of several cancers (breast, colorectal, prostate).
IGF-1 may serve as a diagnostic marker for cancer risk assessment.
mRNA vaccines and the role of mRNA in vaccination
mRNA vaccines educate cells to produce a viral antigen (e.g., the S protein of SARS-CoV-2):
mRNA is delivered to muscle cells, which translate it into the spike protein.
The expressed antigen stimulates the immune system to produce antibodies.
If later infected, these antibodies can help fight the virus.
Resource note: Mayo Clinic overview on different COVID-19 vaccine types.
RNA and transcription-processing biology: summarized references and resources
Key online visuals and readings referenced for RNA biology and translation:
Gene expression and translation resources, including videos on transcription and translation.
Cellular biology references and reviews on PTMs and disease relevance.
Example visual: Human Prion Protein structure (PDB entry 1HJN).
Genetic code and transcription/translation connections: quick reference
Codons and amino acids:
Codons are triplets of bases; there are 64 total codons.
61 codons encode the 20 amino acids; remaining codons include the three stop codons and initiation signals.
Initiation codon: AUG (codes for Methionine).
tRNA and ribosome roles:
tRNA molecules carry specific amino acids and recognize codons via anticodons.
During translation, tRNA delivers amino acids at the ribosome, building the polypeptide chain in the order dictated by the mRNA.
mRNA processing and localization:
Splicing removes introns and joins exons; mature mRNA contains 5' UTR, coding region, and 3' UTR.
5' cap and 3' poly-A tail contribute to stability and translation efficiency.
Summary: core themes tying transcript to phenotype
The flow from genome to phenotype hinges on tightly regulated transcription, RNA processing, translation, folding, and post-translational modifications.
Dysregulation at any stage—signal transduction, RNA processing, protein folding, or PTMs—can lead to disease states, including cancer, neurodegeneration, and infectious disease susceptibility.
Therapeutic strategies (neutralizing antibodies, IGF-1 pathway modulation, mRNA vaccines) leverage understanding of these molecular processes to prevent or treat disease.
Quick reference: key sequences and numbers
Start codon: AUG
Stop codons: UAA, UAG, UGA
Codon length: 3 bases per codon
Total codons: 64
Codons encoding amino acids: 61
5' cap: 7- ext{methylguanine} cap
Poly-A tail length: 50 ext{ to } 250 adenine nucleotides
Core promoter element: TATAAA (TATA box)
Cytosine/Thymine vs Adenine/Guanine: pyrimidines vs purines
Functional motifs: BRE (TFIIB recognition element), Tub promoter orientation, and PIC formation
Note: This condensed set of notes covers the major and minor points from the provided transcript, including specific sequences, process steps, and real-world applications discussed across the slides. For deeper study, consult the indicated sources and the linked figures and reviews in the transcript.