CM08 - Gene Expression and Regulation

What is the central dogma of molecular biology?

DNA → mRNA → Protein. Transcription occurs in the nucleus, translation in the cytoplasm (RER).

What are the structural features of RNA?

Single-stranded with internal base-pairings forming stem loops; contains ribose sugar (unstable, hydrolysis-prone); uracil replaces thymine.

Compare DNA and RNA in terms of structure and properties.

• Sugar: DNA has deoxyribose, RNA has ribose.

• 2’-OH group: absent in DNA, present in RNA.

• Bases: DNA (A,G,C,T), RNA (A,G,C,U).

• Strandedness: DNA usually double, RNA usually single.

• Secondary structure: DNA double helix, RNA many types.

• Stability: DNA stable, RNA easily degraded.

What percentage of RNA is rRNA and what is its function?

~80%. Combines with proteins to form ribosomes, functions as the catalytic subunit (peptidyl-transferase). Four types exist in eukaryotes.

What percentage of RNA is tRNA and what is its function/structure?

~15%. Matches codons to amino acids. Small (80 nt), cloverleaf structure with anticodon loop and acceptor stem (5’-CCA-3’).

What percentage of RNA is mRNA and what is its structure?

~5%. Carries genetic code (monocistronic). Has 5’ cap (7-methylguanosine), coding sequence, and poly(A) tail (20–250 As).

Where are different RNAs located and what are their functions?

• rRNA: cytoplasm, ribosome structure/function.

• mRNA: nucleus/cytoplasm, carries protein code.

• tRNA: cytoplasm, adds AAs to polypeptide.

What are the three eukaryotic RNA polymerases and their products?

RNA Pol I = rRNA (most abundant), Pol II = mRNA (largest), Pol III = tRNA (smallest).

What are features of RNA polymerase activity?

No primer needed, synthesizes 5’→3’, no proofreading, unwinds DNA to copy template strand (antisense).

What are the promoter regions and their elements?

• Core/Basal promoter: TATA-box, Inr (bind general TFs - GTF). This is necessary for the transcription of all genes.

• Proximal promoter: CAAT, GC boxes (bind specific TFs - STF)

• Distal promoter: enhancers/silencers regulate transcription rate far from the active site.

What forms the pre-initiation complex?

Six general transcription factors (GTFs) + RNA Pol II at the TATA-box.

Describe elongation in transcription.

RNA Pol unwinds 10–20 nt at a time, forms transcription bubble, adds ribonucleotides, dsDNA reforms after RNA separates.

What toxin inhibits RNA Pol?

α-amanitin from poisonous mushrooms.

How does transcription terminate?

RNA Pol reaches the AAUAAA polyadenylation signal, continues ~50 nt, pre-mRNA (hnRNA) is then released.

What modifications occur to pre-mRNA before translation?

• 5’ cap (7-methylguanosine) is added — protects from nucleases.

• Poly(A) tail (20–250 As) is added — stabilizes mRNA, aids export; progressively shortened in cytosol.

• Splicing removes introns (GU/AG rule) and joins exons.

What is alternative splicing?

Produces different mature mRNAs from one pre-mRNA based on different combinations of exons joined together.

What is the genetic code and how many codons exist?

The set of codons specifying amino acids. 64 codons: 61 sense, 3 stop (UAA, UAG, UGA), 1 start (AUG).

What are key properties of the genetic code?

Unambiguous (one codon = one AA), degenerate (multiple codons per AA), nonoverlapping/continuous, nearly universal.

What are the three ribosomal sites in translation?

A site = aminoacyl-tRNA, P site = peptidyl-tRNA, E site = exit.

How are tRNAs charged with amino acids?

Aminoacyl-tRNA synthetase activates the AA to aminoacyl-AMP and then attaches it to the 3’-OH of a tRNA acceptor stem. Each AA has a specific synthetase.

What is the wobble hypothesis?

The 3rd codon base allows nontraditional pairing, letting tRNAs recognize multiple codons (degeneracy).

What are the steps of translation initiation?

1. Small ribosomal subunit binds Met-tRNA+IF complex → forms the pre-initiation complex.

2. Complex scans for AUG.

3. AUG pairs with anticodon; methionine = first AA.

4. Met-tRNA placed in P site.

5. Large ribosomal subunit joins; initiation factors released.

What are the steps of elongation?

1. Aminoacyl-tRNA + EF + GTP enters A site.

2. Correct anticodon pairs; GTP hydrolyzed.

3. Peptidyl transferase forms peptide bond between AA in P and A sites.

4. Ribosome translocates: peptidyl-tRNA moves to P, empty tRNA moves to E and exits.

5. Cycle repeats.

How does translation terminate?

Stop codon at A site → release factors bind, polypeptide released, ribosome dissociates.

What are polysomes?

Multiple ribosomes translating the same mRNA simultaneously.

What are the major post-translational modifications?

• Phosphorylation (Tyr, Ser, Thr) → regulation, signaling.

• Glycosylation (O-Ser/Thr, N-Asn).

• Hydroxylation (Pro, Lys in collagen).

• Others: carboxylation, biotinylation, farnesylation.

(Don’t need to know the examples, just the main processes)

How are proteins degraded?

Ubiquitination marks proteins for proteasomal degradation.

What are the two broad purposes of gene expression regulation?

Adaptation (homeostasis) and differentiation/development.

How is gene expression regulated at the DNA level?

• Chromatin accessibility (heterochromatin vs euchromatin).

• Gene amplification (extra copies increase expression, e.g., methotrexate resistance).

• DNA rearrangements (e.g., immunoglobulin recombination).

• Mobile elements (transposons).

How is transcription regulated?

TF binding to promoters, enhancers, silencers; e.g., steroid hormones act via enhancers.

What are the regulatory mechanisms during RNA processing?

• Alternative splicing (e.g., tropomyosin isoforms).

• Alternative polyadenylation (e.g., IgM membrane vs soluble forms).

• RNA editing (e.g., ApoB-48 vs ApoB-100).

• RNA interference (miRNA, siRNA) silencing via binding 3’-UTR.