Chapter 9: Gene Expression, Transcription, and Translation

What is the Central Dogma of Molecular Biology?

Information flows unidirectionally from DNA to Transcription to RNA toTranslation to Protein.

Dr. Archibald Garrod

The "father of chemical genetics"; studied alkaptonuria (a harmless recessive disorder causing urine to turn black upon air exposure) and first proposed that inborn errors of metabolism are due to defective genes affecting specific enzymes.

George Beadle & Edward Tatum

Formulated the "one gene–one enzyme" (now one gene–one protein) hypothesis by exposing bread mold (Neurospora crassa) to UV irradiation and identifying nutritional mutants that required specific exogenous amino acids to grow on minimal media.

What are the properties of the Genetic Code?

• Triplet Codon: Read as non-overlapping blocks of 3 mRNA nucleotides.

• Universal: Shared by virtually all living organisms on Earth.

• Redundant (Degenerate): Multiple codons can specify the same amino acid (61 codons for amino acids, 3 stop codons).

• Not Ambiguous: A single codon never specifies more than one amino acid.

What are the Start and Stop codons?

• Start Codon: AUG (codes for Methionine and establishes the reading frame).

• Stop Codons: UAA, UAG, UGA (do not code for amino acids; terminate translation).

Define Gene Expression and Gene Regulation.

Gene Expression: The process of turning on a gene to transcribe mRNA and translate it into a protein. Gene Regulation: How a cell controls which genes are expressed out of its entire genome.

What are the steps and features of Transcription?

• Location: Nucleus.

• Template: Antisense DNA strand (3' to 5').

• Enzyme: RNA Polymerase II (adds ribonucleotides 5' to 3', does not require a primer, and lacks proofreading activity).

• Steps:

  1. Initiation: Transcription factors bind cis-acting promoter elements like the TATA box to form the transcription initiation complex.

  2. Elongation: RNA polymerase unwinds DNA locally (transcription bubble) and builds pre-mRNA 5' to 3'.

  3. Termination: Stops past the poly-A signal sequence, releasing the pre-mRNA transcript.

What are the three modifications of pre-mRNA processing into mature mRNA?

• 5' Capping: Addition of a modified guanine nucleotide to protect the 5' end from ribonucleases.

• 3' Polyadenylation: Cleavage and addition of a 50–250 adenine nucleotide poly-A tail to protect the 3' end and boost translational efficiency.

• RNA Splicing: Removal of non-coding introns by spliceosomes and joining of coding exons.

What is Alternative RNA Splicing?

The selective joining of different exons from a single pre-mRNA to produce multiple mature mRNA transcript variants (isoforms), allowing about 20,000 genes to encode over 250,000 distinct proteins

What are the components of Translation?

• Location: Cytoplasm (at ribosomes).

• Components: Mature mRNA, Ribosomes (small and large rRNA subunits), Transfer RNA (tRNA), and amino acids

What is the role of tRNA and aminoacyl-tRNA synthetase?

tRNA acts as an adaptor molecule containing a 3' amino acid attachment site and a 5'-anticodon loop that pairs with mRNA codons. Aminoacyl-tRNA synthetase is the enzyme that links/ligates the correct amino acid to its corresponding tRNA using ATP

What is Wobble Base Pairing?

Flexible (non-Watson-Crick) base pairing permitted between the 3' base of an mRNA codon and the 5' base of a tRNA anticodon, explaining why fewer than 45 tRNAs exist for 61 sense codons.

What are the three ribosomal sites during Translation?

• A site (Aminoacyl): Binds the incoming charged tRNA carrying the next amino acid.

• P site (Peptidyl): Holds the tRNA attached to the growing polypeptide chain (initiator Met starts here).

• E site (Exit): Where uncharged tRNAs exit the ribosome.

Describe the steps of Translation (Initiation, Elongation, Termination).

• Initiation: Small ribosomal subunit binds mRNA, initiator tRNA ({Met}, anticodon UAC) binds the start codon ({AUG}), and the large subunit joins to form the initiation complex.

• Elongation: Codon recognition in the A site, peptide bond formation (catalyzed by rRNA acting as a ribozyme), and translocation shifting tRNAs from A to P and P to E.

• Termination: A stop codon enters the A site, a release factor binds instead of tRNA, releasing the completed polypeptide and dissociating the ribosome.

What are Polysomes?

Multiple ribosomes simultaneously translating a single mRNA molecule, greatly amplifying protein synthesis.

What is the structure of an Amino Acid?

Central alpha-carbon bonded to a Hydrogen, an Amino group ({NH}2 /{NH}3+), a Carboxyl group ({COOH}/{COO-}), and a variable R-group (side chain) that dictates its chemical category (nonpolar, polar, uncharged, charged acidic/basic).

What is a Peptide Bond?

A covalent bond formed by a dehydration/condensation reaction between the carboxyl group of one amino acid and the amino group of the next, linking amino acids into a polypeptide chain with an N-terminus and C-terminus.

What are the Four Levels of Protein Structure?

• Primary Structure: The linear, ordered sequence of amino acids linked by peptide bonds.

• Secondary Structure: Local folding into $\alpha$-helices and $\beta$-pleated sheets stabilized entirely by backbone hydrogen bonds (R-groups are not involved).

• Tertiary Structure: The overall 3D conformation of a single polypeptide chain driven by interactions between R-groups (hydrophobic interactions, ionic bonds, hydrogen bonds, and covalent disulfide bridges between cysteines).

• Quaternary Structure: The association of two or more polypeptide chains (e.g., Hemoglobin has 4 chains; Collagen has 3).

What are examples of diseases caused by protein misfolding?

• Alzheimer's Disease: Misfolded beta-amyloid proteins form toxic insoluble aggregates (plaques) and neurofibrillary tangles.

• Cystic Fibrosis: Defect in folding the CFTR protein (chloride ion transporter), resulting in thick, sticky mucus.

• Creutzfeldt-Jakob Disease (Mad Cow): Caused by prions—infectious misfolded proteins that convert normal conformers into rogue disease-causing conformations

What are the four main mechanisms of Gene Regulation in Eukaryotes?

• Transcriptional Regulation: Chromatin remodeling and transcription factor binding control whether a gene is transcribed.

• Post-Transcriptional Regulation: Alternative splicing, mRNA stability, and RNA interference (RNAi) via microRNAs regulate mRNA availability.

• Translational Regulation: Poly-A tail length and UTRs control translation rates.

• Post-Translational Regulation: Protein chemical modifications (phosphorylation, ubiquitination, acetylation, etc.) and protein turnover control active protein availability.

Heterochromatin vs. Euchromatin

• Heterochromatin: Tightly compacted, heavily stained, transcriptionally inactive chromatin (subdivided into telomeric, centric, and intercalary).

• Euchromatin: Loosely condensed, lightly stained, transcriptionally active chromatin containing most active genes.

Epigenetic modifications: Histone Acetylation vs. DNA Methylation

• Histone Acetylation: Addition of acetyl groups to lysine residues by Histone Acetyl Transferases (HATs) neutralizes positive charges, relaxes chromatin into euchromatin, and turns genes ON (Deacetylation by HDACs compacts chromatin and turns genes OFF).

• DNA Methylation: Addition of methyl groups to cytosine bases by DNA methyltransferases represses transcription by blocking transcription factor binding or recruiting repressors, turning genes OFF.

What is RNA interference (RNAi)?

Gene silencing technology where small non-coding microRNAs bind target mRNA; 100% complementary base pairing leads to mRNA degradation, while partial pairing leads to transcriptional/translational repression

Cis-acting elements vs. Trans-acting factors

• Cis-acting elements: Non-coding DNA regulatory sequences on the same chromosome (e.g., TATA box, CAAT box, GC box, enhancers).

• Trans-acting factors: Diffusible regulatory proteins (transcription factors, activators, repressors) that bind to cis-acting elements to control transcription.