Lec 3+4

Prokaryotic vs. Eukaryotic Cis-Regulatory Element Positioning

Prokaryotic regulatory elements are typically close to the transcription start site. In contrast, eukaryotic regulatory elements can be far upstream, far downstream, or within introns.

Bacterial Activation over Long Distances (NtrC Looping)

The bacterial activator protein NtrC binds to a distant cis-regulatory sequence and directly contacts RNA polymerase by causing the intervening DNA to loop out, activating transcription.

Bacteriophage Lambda Pathways

Bacteriophage Lambda infects bacteria and circularizes its DNA. It chooses between two lifestyles: the prophage pathway (integrates viral DNA into host chromosome to replicate during host cell division) or the lytic pathway (synthesizes viral proteins, rapidly replicates, packages new viruses, and lyses host cell).

Bacteriophage Lambda Switch: Key Regulators

The switch between prophage and lytic states is controlled by two gene regulatory proteins that mutually repress each other's synthesis: Lambda repressor protein (cI) and Cro protein.

Lambda Switch: Prophage State (cI Active)

Lambda repressor (cI) occupies the operator, which blocks synthesis of Cro and activates its own synthesis via a positive feedback loop, keeping most viral DNA silenced.

Lambda Switch: Lytic State (Cro Active)

Cro protein occupies the operator, which blocks synthesis of Lambda repressor and permits its own synthesis, leading to extensive viral transcription, DNA replication, packaging, and host cell lysis.

Lambda Switch Trigger Mechanism

The switch from prophage to lytic state is triggered by the host cell's response to DNA damage, which inactivates the Lambda repressor. Under good growth conditions, Lambda repressor maintains the prophage state.

Positive Feedback Loop & Cell Memory

Protein A activates its own transcription. An initial transient signal turns on Gene A, and because A continues to transcribe itself in daughter cells, it creates cell memory.

Negative Feedback Loop

Protein A represses its own transcription, serving to limit protein production and stabilize expression levels.

Flip-Flop Device

An indirect positive feedback loop where Protein A represses Gene B, and Protein B represses Gene A. The Cro/Lambda repressor switch is a classic natural example.

Feed-Forward Loop Function

Transcription factor A activates transcription factor B, and both A and B are required to transcribe target Gene Z. This measures signal duration and filters out noise because a brief input does not allow enough B to accumulate, whereas a prolonged input enables Z transcription.

Synthetic Biology & The Repressilator

Synthetic biology constructs artificial circuits. The Repressilator is a synthetic gene oscillator built from a delayed negative feedback loop using three repressors: Lac repressor (A), Tet repressor (B), and Lambda repressor (C).

Repressilator Mechanism & Outcome

Repressor A represses B, B represses C, and C represses A. This produces regular, delayed negative feedback oscillations in gene expression, as confirmed experimentally using fluorescently tagged proteins.

Transcription Attenuation Mechanism

A mechanism in prokaryotes and eukaryotes where transcription is prematurely terminated because the nascent RNA folds into a secondary structure that interferes with RNA polymerase.

Riboswitches Definition

Short RNA sequences (found in prokaryotes, plants, and some fungi) that directly bind a small molecule ligand, causing a conformational change that regulates gene expression.

Purine Riboswitch: Low Guanine State

When guanine levels are low, the riboswitch structure allows RNA polymerase to continue transcribing through the 5' UTR into coding sequences, keeping purine biosynthetic genes ON.

Purine Riboswitch: High Guanine State

When guanine levels are high, guanine binds the riboswitch, triggering a conformational change that forms a transcription terminator, causing RNA polymerase to abort transcription so purine genes are OFF.

The Three Eukaryotic RNA Polymerases

RNA Pol I transcribes 5.8S, 18S, and 28S rRNAs genes. RNA Pol II transcribes all protein-coding genes, miRNAs, siRNAs, lncRNAs, snoRNAs, and most snRNAs. RNA Pol III transcribes tRNAs, 5S rRNA, and small nuclear RNAs. (The bigger the S value, the more RNA is present in the molecule's sedimentation during centrifugation, indicating its size.)

General Transcription Factors (GTFs) Definition

A set of conserved proteins required by RNA Polymerase II to locate eukaryotic promoters, unwind DNA, and initiate transcription.

TFIID Structure & Function

TFIID contains TBP (TATA-binding protein) and 11 TAFs (TBP-associated factors). It recognizes and binds the TATA box and other promoter sequences near the start site.

TFIIB, TFIIA, and TFIIF Functions

TFIIB recognizes the BRE promoter element and accurately positions RNA Pol II. TFIIA stabilizes TFIID binding. TFIIF stabilizes RNA Pol II's interaction with TFIIB and recruits TFIIE and TFIIH.

TFIIE and TFIIH Functions

TFIIE recruits and regulates TFIIH. TFIIH contains 10 subunits and functions as a helicase (unwinds promoter DNA) and a kinase (phosphorylates Ser5 on the C-terminal domain / CTD of RNA Pol II to release it from the promoter).

Mediator Complex Role

A massive protein complex that serves as an essential intermediate/bridge, transmitting signals between upstream gene regulatory proteins (activators/repressors) and RNA Polymerase II / GTFs.

Modular Design of Eukaryotic Activators

Eukaryotic activators feature a modular architecture consisting of two independent domains: a DNA-binding domain (DBD) (recognizes specific DNA sequences) and an Activation domain (AD) (accelerates transcription rate).

Chimeric Activator Experiment (Gal4/LexA)

Because DBDs and ADs are modular, swapping the Gal4 DBD with the LexA DBD creates a functional chimeric protein that activates transcription specifically at LexA operator sequences.

Nucleosome Core Particle Structure

The basic unit of eukaryotic chromatin, consisting of 147 bp of DNA wrapped 1.67 (1 1/3) times around a core histone octamer (two copies each of H2A, H2B, H3, and H4).

Linker DNA & Histone H1

Linker DNA (~10–80 bp) connects adjacent nucleosomes. Histone H1 acts as a clip holding DNA wrapped around the nucleosome core together.

Chromatin Fiber Models

Nucleosomes pack into compact higher-order chromatin fibers described by either the Zigzag model or the Solenoid model. Tightly packed chromatin physically prevents transcription machinery assembly.

Four Major Ways Activators Alter Chromatin

Activators increase promoter accessibility by: 1) Nucleosome sliding, 2) Histone removal, 3) Histone variant replacement, and 4) Specific patterns of histone modification.

Nucleosome Sliding Mechanism

Activators recruit ATP-dependent chromatin remodeling complexes that use ATP hydrolysis to slide nucleosomes along DNA, exposing covered promoter sequences.

Histone Removal & Replacement

Remodeling complexes work alongside histone chaperones to completely evict nucleosome octamers or replace standard histones with looser-binding histone variants (e.g., variant H2A-H2B dimers).

Histone Modifications & "Writer" Enzymes

Covalent modifications occur on N-terminal tails of core histones. Histone acetyltransferases (HATs) add acetyl groups, histone kinases add phosphate groups, and histone methyltransferases add methyl groups.

Histone Code "Readers"

Specialized reader proteins recognize distinct combinations of histone tail modifications and bind them to execute specific biological meanings.

Key Histone H3 Modification Meanings

H3K9 methylation: Heterochromatin formation & gene silencing.
H3K4 methylation + H3K9 acetylation: Active gene expression.
H3S10 phosphorylation + H3K14 acetylation: Active gene expression.
H3K27 methylation: Hox gene silencing & X-inactivation.

Human Interferon Activation: Steps 1 & 2

Step 1: Activator protein binds chromatin DNA and recruits a Histone Acetyltransferase (HAT).
Step 2: HAT acetylates H3K9 (lysine 9 of H3) and H4K8 (lysine 8 of H4).

Human Interferon Activation: Steps 3 & 4

Step 3: Activator recruits a histone kinase (HK).
Step 4: HK phosphorylates H3S10 (serine 10 of H3) — this step strictly requires prior H3K9 acetylation.

Human Interferon Activation: Steps 5 & 6

Step 5: H3S10 phosphorylation signals HAT to acetylate H3K14.
Step 6: TFIID and a chromatin remodeling complex bind modified tails to initiate transcription.

Prokaryotic vs. Eukaryotic Repression

Prokaryotic repressors directly compete with RNA polymerase for promoter binding. Eukaryotic repressors rarely compete with RNA polymerase directly and instead use diverse mechanisms to inhibit expression.

Repression via Competitive DNA Binding & Surface Masking

Competitive binding: Repressor binding site overlaps activator site, physically blocking activator binding.
Masking activation surface: Repressor binds adjacent to activator and interacts with its activation domain, preventing interaction with transcription machinery.

Repression via Direct GTF Interaction & Remodeling

Direct GTF interaction: Repressor directly contacts general transcription factors (e.g., TFIID) to halt initiation.
Chromatin remodeling recruitment: Repressor recruits remodeling complexes that re-wrap nucleosomes into compact structures.

Repression via Histone Modifications (HDACs & Methyltransferases)

Repressors recruit histone deacetylases (HDACs) to strip acetyl groups from histone tails, or recruit histone methyltransferases to write repressive methyl marks.

Spreading Chromatin Marks (Reader-Writer Complexes)

A regulatory protein recruits a writer enzyme to modify a histone. A code-reader protein binds that mark and recruits another writer, propagating the repressive histone mark along chromatin until reaching a barrier sequence.

DNA Methylation & Heterochromatin Stabilization

Reader-writer complexes attract DNA methylase, which methylates nearby cytosine bases in DNA. DNA methyl-binding proteins bind methylated cytosines and lock chromatin into a stable, repressed structure.

Epigenetic Inheritance Definition

Heritable changes in gene expression and chromatin state that are passed on to daughter cells without altering the underlying DNA sequence (e.g., via DNA methylation and histone marks).