From Gene to Protein

Contribution of Metabolic Defects to Gene-Protein Understanding

• Metabolic defect studies, especially genetic diseases like alkaptonuria, linked specific genes to enzymes in metabolic pathways.
• Observable phenotypic consequences of enzyme absence/malfunction emphasized gene encoding for proteins.

Beadle and Tatum Experiment

• Used Neurospora crassa exposed to X-rays, identifying mutants requiring specific nutrients.
• Each mutant had a single gene defect linked to a missing enzyme, leading to the "one gene–one enzyme" hypothesis.

One Gene – One Enzyme Hypothesis Revision

• Not all proteins are enzymes; some are multi-polypeptide.
• Single genes can produce multiple proteins via alternative splicing.
• Modern understanding: "one gene–one polypeptide."

Transcription and Translation Basics

• Transcription: DNA to mRNA via RNA polymerase in nucleus.
• Translation: mRNA to polypeptide at ribosome, tRNAs bring amino acids.

Prokaryotic vs. Eukaryotic Transcription and Translation

• Location: Prokaryotes in cytoplasm, eukaryotes transcription in nucleus, translation in cytoplasm.
• RNA Processing: Minimal in prokaryotes; extensive in eukaryotes.
• Timing: Concurrent in prokaryotes, separated in eukaryotes.
• Gene Organization: Operons in prokaryotes, monocistronic mRNA in eukaryotes.
• RNA Polymerases: One type in prokaryotes, three types in eukaryotes.

Genetic Code Encoding Amino Acids

• DNA sequences of three-nucleotide codons encode amino acids.
• Each mRNA codon specifies an amino acid or stop signal during translation.

Genetic Code Redundancy

• Redundant: Multiple codons for most amino acids.
• Not Ambiguous: Each codon specifies only one amino acid.

Molecular Components of Transcription

• DNA template strand, RNA polymerase, transcription factors, promoter region, mRNA.

Enhancers and Promoters

• Promoters: DNA sequences for RNA polymerase binding.
• Enhancers: Distal elements binding activators to increase transcription.

RNA Modification in Eukaryotic Cells

• 5' capping: Modified guanine added at 5' end.
• 3' polyadenylation: Poly-A tail added at 3' end.
• Splicing: Removal of introns, joining exons.

RNA Splicing Process

• Conducted by spliceosome (snRNAs and proteins).
• Recognizes splice sites, cuts out introns, joins exons.

From Gene to Protein II

Introns' Functional and Evolutionary Importance

• Functional: Alternative splicing, gene regulation, mRNA transport/stability.
• Evolutionary: Facilitate exon shuffling, increasing genomic flexibility.

tRNAs in Polypeptide Elongation

• tRNAs bring correct amino acids, pairing anticodons with mRNA codons.
• Elongation involves tRNA entry, peptide bond formation, tRNA exit.

tRNA Pairing with mRNA Codons

• Anticodon loop pairs complementary and antiparallel to mRNA codon.

Ribosomes in Protein Synthesis

• Bind mRNA, recruit tRNAs, catalyze peptide bonds, and move along mRNA.

Protein Synthesis Termination

• Stop codon reached, release factor binds, triggers polypeptide release.

Polypeptide Shuttle to ER

• Signal peptides direct proteins to ER, SRP halts translation and docks at ER.

Types of DNA Mutations

• Point mutations: Substitution, insertions/deletions, duplication, inversion, translocation.

Silent, Missense, and Nonsense Mutations

• Silent: No protein change.
• Missense: Different amino acid, potential functional impact.
• Nonsense: Premature stop, truncated protein.

Prokaryotic Gene Expression

Bacterial Enzyme Production Regulation

• Operons regulate at transcriptional level, responding to environmental changes.
• Negative Regulation: Repressor blocks transcription.
• Positive Regulation: Activator enhances transcription.
• Feedback Inhibition: Pathway end product inhibits enzyme/gene expression.

Operon Components

• Promoter, operator, structural genes, regulatory gene.

Repressor Function

• Binds operator to block transcription, active in inducible/repressible systems.

Repressible vs. Inducible Operons

• Inducible: Normally off, active by substrate.
• Repressible: Normally on, deactivated by product.

Eukaryotic Gene Expression

Eukaryotic Gene Expression Regulation Stages

• Chromatin accessibility, transcriptional control, RNA processing, mRNA transport, stability, translation control, post-translational modification, protein degradation.

Epigenetic Inheritance and Chromatin Modification

• Heritable changes without DNA sequence change, including histone modifications, DNA methylation.

Transcription Factors

• Bind DNA, help/hinder RNA polymerase, act as activators/repressors.

Gene Activation in Liver vs. Lens Cells

• Differentiation through transcription factors, epigenetic differences.

Alternative RNA Splicing

• Allows multiple protein variants, increases diversity, enables tissue-specific expression.

mRNA and Protein Lifespan Determinants

• mRNA: 3' UTR sequences, poly-A tail length, regulatory proteins/miRNAs.
• Proteins: Degradation signals, proteasome targeting.

Non-Coding RNAs in Gene Expression

• miRNAs: Block translation/degrade mRNA.
• siRNAs: RNA interference role.
• lncRNAs: Chromatin remodeling, transcription regulation, splicing.

Cytoplasmic Determinants in Development

• Unevenly distributed in egg, leading to differential gene expression post-fertilization.

MyoD as Gene Expression Regulator

• Activates muscle-specific genes, converts non-muscle to muscle-like cells.

Pattern Formation in Fruit Fly

• Sequential gene activation establishes body plan: maternal, gap, pair-rule, segment polarity, homeotic genes.

bicoid Gene in Axis Establishment

• mRNA localized at egg's anterior, forms protein gradient post-fertilization.

Cancer-Related Genes

• Oncogenes: Promote uncontrolled division.
• Tumor Suppressors: Inhibit growth/repair DNA.

Colorectal Cancer Mutation Pathway

• Multi-step: APC loss, ras activation, DCC/p53 loss.

Biotechnology

Gene Cloning in Bacteria

• Steps: Isolate, insert into plasmid, transform bacteria, select, grow.

Restriction Enzymes in DNA Cloning

• Cut DNA at specific sites, creating recombinant DNA with sticky/blunt ends.

Genomic vs. cDNA Libraries

• Genomic: Includes all DNA.
• cDNA: From mRNA, no introns.

Screening Libraries for Genes

• Hybridization: Labeled probe.
• PCR: Specific primers.
• Antibody: Detect protein.

Southern Blot Analysis Steps

• Digest, gel electrophoresis, blotting, hybridize, detect.

RFLP for DNA Comparison

• Fragment length differences reflect mutations, used in mapping, forensics.

Expression System Selection Factors

• Host organism, post-translational modifications, folding/solubility, cost/time, safety.

PCR Amplification

• Cycles of denaturation, annealing, extension for DNA duplication.

DNA Sequencing with Dideoxyribonucleotides

• Sanger method: Reactions with ddNTPs terminate at specific bases, sequence from lengths.

Northern Blot Analysis Purpose

• Detect RNA, assess expression, compare across conditions.

Reverse-Transcriptase PCR

• Converts mRNA to cDNA, then amplifies.

DNA Microarray Analysis Purpose

• Gene expression patterns, disease profiling, differential expression.

Determining Gene Function and Knockdown

• Knockout, RNAi, overexpression, CRISPRi.

SNPs in Gene Location

• Linked to disease traits through GWAS, pinpoint gene loci.

Animal Cloning Methodology

• Somatic cell nuclear transfer steps.

Dolly the Sheep's Early Death

• Shortened telomeres, epigenetic issues, immune/organ function.

Stem Cell Disease Treatment

• Differentiate/replace tissues, regenerative medicine, iPS cells.

Therapeutic Gene Delivery

• Viral/non-viral methods, CRISPR-based editing.