Final Comprehensive Study Notes

Gene Regulation in Prokaryotic Cells and Eukaryotic Transcription

The Central Dogma (Extended Version)

• Classic Flow of Genetic Information: DNA replication $\rightarrow$ Transcription $\rightarrow$ Translation.

• Extended Version: Includes Reverse Transcription (RNA to DNA).

Regulation of the Formation of Open-Promoter Complexes

• Nucleotide Triphosphate (NTP) Concentrations:

  • High initiating [NTP], specifically ATP and GTP (purines), stabilizes the Open-Promoter Complex.

  • pppGpp and ppGpp (Guanosine Tetraphosphate): These function as "alarmones" produced when cells are starved for amino acids.

  • Mechanism of Alarmone Production: Uncharged tRNAs bind to the A site on ribosomes. The RelA protein senses this signal and produces the alarmones.

  • Effect on rRNA Genes: The presence of pppGpp and ppGpp destabilizes the open promoter complex at rRNA gene promoters.

Prokaryotic Transcription Initiation

• Abortive Transcripts: Short RNA sequences ranging from $2-6\text{ nt}$ produced during the initial phase of transcription.

• Promoter Clearance: Occurs after the synthesis of $9$ or $10\text{ nt}$. High initiating [NTP] stabilizes this transition.

The Lac Operon: Structural and Genetic Components

• B-galactosidase (lacZ): An enzyme that breaks the B-galactosidic bond in lactose to produce Galactose and Glucose. It also produces Allolactose, which acts as the natural inducer.

• Permease (lacY): A transport protein that moves lactose into the cell. Mutants lacking this (lacY-) are called "cryptic" because they cannot grow on lactose but show activity in lysed cells.

• Galactoside Transacetylase (lacA): A protein of previously unknown function involved in the operon.

• Regulatory Elements:

  • lacI: Encodes the Repressor protein, which is an allosteric protein forming a tetramer.

  • Operator (O): The DNA binding site for the repressor.

  • Polycistronic mRNA: One promoter controls the transcription of multiple genes (lacZ, lacY, lacA), each with its own Shine-Dalgarno (SD) sequence.

Regulatory Mutations and Merodiploids

• Constitutive Mutants: Mutants where the operon is always "on."

  • Operator Constitutive ($O^c$): Mutations in the operator that prevent repressor binding. This is cis-dominant, affecting only the genes on the same DNA strand.

  • Repressor Mutation (lacI-): Mutations in the repressor gene. These are trans-dominant because the repressor protein is a diffusible factor that can bind to operators on different DNA molecules.

• Phenotypes:

  • Uninducible: Operon cannot be turned on.

  • Unrepressible: Operon cannot be turned off (constitutive).

Mechanisms of lac Repressor Action

• Model 1 (Mutual Exclusion): The repressor prevents the binding of RNA polymerase (RNAP) to the promoter. Recent data from Record et al. support this competition hypothesis.

• Model 2 (Promoter Clearance Inhibition): The repressor allows RNAP to bind and form an open promoter complex but inhibits the transition from abortive transcription to processive transcription (blocking promoter clearance).

• Experimental Evidence for Model 2:

  • Pastan et al.: Observed rifampicin-resistant transcription in the presence of repressor once inducer (IPTG) was added. Rifampicin only blocks the first phosphodiester bond formation; if an open complex exists, transcription proceeds.

  • Lee and Goldberg: Performed run-off experiments showing RNAP forms an open complex even with the repressor present.

  • Heparin Assay: Heparin blocks new initiation. The formation of heparin-resistant complexes in the presence of the repressor suggests an open promoter complex was formed.

Catabolite Activator Protein (CAP) and cAMP

• Glucose Effect: When glucose is present, cAMP levels drop because $IIA^{Glc}$ inhibits adenylate cyclase.

• Catabolite Repression: Cells prefer glucose. If glucose is high and lactose is high, the operon transcription is minimal to avoid wasting energy making glucose.

• CAP-cAMP Complex: A positive regulator.

  • Low glucose $\rightarrow$ High cAMP $\rightarrow$ CAP-cAMP binds the CAP binding site ($TGTGA$).

  • Mechanism: CAP facilitates the formation of the open promoter complex by increasing the association constant $K_B$. It interacts with the $\alpha\text{-CTD}$ (Carboxyl Terminal Domain) of RNAP.

• DNA Bending: CAP-cAMP binding induces a bend in the DNA of approximately $90$ to $100$ degrees (confirmed by X-ray crystallography and gel mobility assays).

Molecular Biology Applications: Blue-White Selection

• Cloning Vectors (e.g., pUC18, pUC19): Contain a Multiple Cloning Site (MCS) within the lacZ gene.

• X-gal/IPTG:

  • Blue Colonies: No insert; lacZ is functional, cleaving X-gal.

  • White Colonies: Insert present; lacZ is disrupted.

• Insertion Types:

  • In-frame: Multiples of $3$.

  • Out-of-frame: Causes a frameshift (1 or 2 + multiples of 3). Multiples of 3 and <~450\text{ bp} might still allow blue colonies if the insertion doesn't destroy activity.

Eukaryotic RNA Polymerases

• Pol I: Located in the nucleolus; synthesizes rRNA genes (high CG content).

• Pol II: Located in the nucleoplasm; synthesizes mRNA and miRNAs.

• Pol III: Located in the nucleoplasm; synthesizes small RNAs like $5S\text{ rRNA}$ and $tRNA$.

• Identification: Separated by DEAE-Sephadex chromatography.

• Toxin Sensitivity ($\alpha$-amanitin):

  • Pol II: Extremely sensitive (I_{50} = 0.02\,g/mL).

  • Pol III: Moderately sensitive (I_{50} = 20\,g/mL).

  • Pol I: Insensitive.

Eukaryotic Promoter Elements

• Core Promoter: Includes the TATA box ($TATA(T/A)AA(G/A)$), Initiator (Inr) ($YYAN(T/A)YY$), BRE (TFIIB recognition element), DCE (Downstream Core Element), and DPE (Downstream Promoter Element).

• Proximal Promoter Elements: Located $37$ to $250\text{ bp}$ upstream (e.g., GC box, CCAAT box).

• Enhancers and Silencers: Cis-acting elements that are orientation- and position-independent.

  • Silencers: Interact with repressors and can cause chromatin to condense.

Mapping Transcription Start Sites

• S1 Mapping: Uses S1 nuclease to digest single-stranded DNA/RNA. Used to show that the TATA box is crucial for locating the start of transcription.

• Primer Extension: Uses reverse transcriptase and a labeled primer to determine the exact $5'$ end of a transcript.

General Transcription Factors and the Preinitiation Complex (PIC)

• Assembly Order: TFIID $\rightarrow$ TFIIA $\rightarrow$ TFIIB $\rightarrow$ TFIIF + Pol II $\rightarrow$ TFIIE $\rightarrow$ TFIIH.

• TFIID Structure: Composed of TBP (TATA-box Binding Protein) and TAFs (TBP-associated factors).

  • TBP: Saddle-shaped protein that binds the minor groove of the TATA box, bending the DNA $80$ degrees. It is used by all three RNA polymerases.

  • TAFs: Can serve as coactivators, histone acetyltransferases (HATs), or kinases.

• TFIIH Functions:

  • Kinase Activity: Phosphorylates the CTD of RNA Polymerase II (specifically Ser5 for promoter clearance).

  • Helicase Activity: Uses ATP to unwind DNA and expand the transcription bubble. RAD25 is a crucial subunit for this activity.

The Elongation Factor TFIIS

• Transcription Arrest: Occurs when RNAP backtracks too far at defined pause sites.

• TFIIS Action: Stimulates the inherent RNase activity of RNAP II to cleave off misincorporated or stalled nucleotides, allowing elongation to resume and improving proofreading.

Classes of DNA-Binding and Dimerization Domains

• DNA-Binding Motifs:

  • Zinc Fingers: $C_2H_2$ type (e.g., Zif268) and $C_4$ type (Nuclear receptors like the Glucocorticoid receptor).

  • Homeodomains (HD): Follow a helix-turn-helix structure with an additional N-terminal arm that binds the minor groove.

  • bZIP and bHLH: Contain highly basic regions for DNA binding linked to dimerization motifs.

• Dimerization Domains: Leucine Zipper, Coiled Coil, and Helix-Loop-Helix.

• Activation Domains: Classified as Acidic (GAL4), Glutamine-rich (Sp1), or Proline-rich (CTF).

Recruitment Models in Eukaryotes

• Model 1 (Stepwise): Preinitiation complex is built protein by protein.

• Model 2 (Holoenzyme Recruitment): An intact holoenzyme (Pol II + several TFs) is recruited to the promoter as a single unit.

• Evidence: VP16 binds TFIID (supports recruitment). Ptashne's work with GAL11P suggested that any contact between an activator and any part of the holoenzyme can trigger recruitment. However, Kornberg found unequal stoichiometry of components, arguing against the universal delivery of a pre-assembled holoenzyme.

Questions & Discussion

• Q: Which of the following technique(s) is used to map transcription start sites?

• A: S1 Mapping and Primer Extension are the primary methods discussed for identifying the specific nucleotide where transcription begins.