lecture 1-3: prokaryotic gene regulation

Comprehensive vocabulary flashcards covering the core mechanisms of bacterial transcriptional regulation, RNA polymerase structure, operons, two-component systems, global regulons, termination pathways, attenuation, and riboswitches from Lecture 1–3.

Efficient Economy

The principle of gene regulation ensuring that cells produce proteins in the correct quantities to avoid placing a metabolic burden on the cell and wasting energy and raw materials.

Environmental Responsiveness

The regulatory ability of a bacterium to detect fluctuating external conditions (such as nutrient availability) and rapidly adjust gene expression to match demand.

Internal State Responsiveness

The regulatory mechanism by which bacterial cells monitor and respond to intracellular cues, such as metabolite concentrations, preventing unnecessary synthesis even when substrates are present.

Coordination (Gene Regulation)

The simultaneous expression of groups of genes whose protein products contribute to the same biological process, often achieved by organising genes into operons.

RNA Polymerase Core Enzyme

The multi-subunit bacterial enzyme complex ($\alpha_2\beta\beta'\omega$) capable of RNA synthesis but lacking the ability to recognise specific promoter sequences on its own.

RNA Polymerase Holoenzyme

The complete, active bacterial transcription complex composed of the core enzyme ($\alpha_2\beta\beta'\omega$) associated with a sigma ($\sigma$) factor that directs promoter recognition.

$\alpha\text{CTD}$ (C-Terminal Domain of the $\alpha$ Subunit)

The C-terminal domain of the RNA polymerase $\alpha$ subunit that interacts directly with DNA UP elements and transcriptional activator proteins to stabilise RNAP binding.

$\beta$ and $\beta'$ Subunits

The catalytic core and 'crab claw' structure of RNA polymerase; the $\beta$ subunit catalyses nucleotide addition, while $\beta'$ coordinates $\text{Mg}^{2+}$ for phosphodiester bond chemistry.

$\omega$ (Omega) Subunit

The smallest subunit of bacterial RNA polymerase that functions as an assembly chaperone to help the $\beta'$ subunit fold properly.

Sigma ($\sigma$) Factor

A bacterial transcription initiation factor that binds to the core RNA polymerase enzyme, conferring specificity for promoter sequence recognition.

Housekeeping Sigma Factor ($\sigma^{70}$)

The primary sigma factor in E. coli encoded by rpoD, which mediates basal transcription by recognising the $-35$ ($\text{TTGACA}$) and $-10$ ($\text{TATAAT}$) promoter motifs separated by an optimal spacer of $\sim 17\,\text{bp}$.

Constitutive Promoter

A promoter whose transcriptional activity relies solely on recognition by the RNA polymerase holoenzyme without requiring additional regulatory proteins to switch between expression states.

Closed Complex

The initial intermediate in transcription initiation where the RNA polymerase holoenzyme recognises and binds promoter DNA while the DNA remains completely double-stranded.

Open Complex Formation (Isomerization)

The stage of transcription initiation where approximately $13\,\text{bp}$ of DNA are locally melted around the transcription start site, forming a single-stranded transcription bubble that exposes the template strand.

Abortive Initiation

The process during early transcription initiation in which RNA polymerase synthesises short RNA transcripts of a few nucleotides and repeatedly releases them without escaping the promoter.

Promoter Escape

The event during transcription initiation occurring once a transcript reaches approximately $10\,\text{nt}$, prompting RNA polymerase to undergo a conformational change, release the sigma factor, and clear the promoter.

Elongation (Bacterial Transcription)

The phase of transcription where RNA polymerase travels along the template DNA, maintaining the transcription bubble, while its rudder and lid domains separate the newly synthesised RNA from the DNA template.

Operator

A specific DNA sequence recognised and bound by a transcription factor, typically positioned near or overlapping a promoter to regulate transcription initiation.

Helix-Turn-Helix Motif

A common bacterial DNA-binding protein motif composed of two $\alpha$-helices separated by a short turn, where one recognition helix establishes sequence-specific contacts in the major groove of DNA.

Steric Hindrance (Repression)

A mode of transcriptional repression where a repressor protein binds to an operator that physically overlaps the promoter, preventing RNA polymerase from accessing the DNA.

DNA Looping (Repression)

A mechanism of transcriptional repression where a repressor simultaneously binds two non-contiguous operator sites, looping the intervening DNA to stably block transcription.

Operon

A unit of bacterial genomic DNA containing a cluster of functionally related genes transcribed together under the control of a single shared promoter into a single polycistronic mRNA.

lacZ

A structural gene in the lac operon encoding $\beta$-galactosidase, an enzyme that cleaves lactose into glucose and galactose and isomerises lactose into allolactose.

lacY

A structural gene in the lac operon encoding lactose permease, a membrane-bound transporter that facilitates lactose uptake into the bacterial cell.

lacA

A structural gene in the lac operon encoding transacetylase, an enzyme with a minor auxiliary role associated with detoxification.

LacI (Lac Repressor)

A homotetrameric transcription factor that binds simultaneously to the proximal operator and one of two distal operators of the lac operon, repressing transcription via DNA looping.

Allolactose

A metabolic isomer of lactose that functions as an inducer of the lac operon by binding the regulatory domain of LacI, triggering a conformational change that prevents DNA binding.

Leaky Expression

The low basal level of transcription that persists even when a promoter is in a repressed state, ensuring that small quantities of essential enzymes (such as permease and $\beta$-galactosidase) are available to sense inducers.

Negative Autoregulation

A gene network motif in which a transcription factor represses its own promoter, promoting faster response times and stabilising protein concentrations against fluctuations.

Positive Autoregulation

A gene network motif where a transcription factor activates its own transcription, creating a positive feedback loop that can establish bistability.

Bistability

The capacity of a gene regulatory circuit to stably maintain either of two distinct, persistent expression states ('on' or 'off') without spontaneously switching between them.

Catabolite Repression

The regulatory repression of genes encoding pathways for alternative carbon sources when a preferred carbon source, such as glucose, is present.

Cyclic AMP (cAMP)

A small intracellular signaling molecule synthesised from ATP by adenylate cyclase; its cellular concentration rises when glucose is depleted and decreases when glucose transport is active.

CRP (cAMP Receptor Protein)

A global transcriptional activator (also known as CAP) that binds cAMP, undergoes a conformational change, and binds promoter DNA to recruit RNA polymerase via contact with $\alpha\text{CTD}$.

Class I Promoter (CRP Activation)

A promoter architecture (such as $P_{\text{lac}}$) where a CRP dimer binds upstream of the core promoter elements and makes contact exclusively with the $\alpha\text{CTD}$ of RNA polymerase.

Class II Promoter (CRP Activation)

A promoter where CRP binds at a target site overlapping the $-35$ element, contacting both the $\alpha\text{CTD}$ and the $\sigma$ subunit of RNA polymerase directly.

Class III Promoter (CRP Activation)

A promoter where transcription activation requires the cooperative binding of multiple CRP dimers or CRP acting cooperatively alongside another transcription activator.

Regulon

A global regulatory collection of non-contiguous genes or operons scattered across the bacterial genome that are co-regulated by a shared transcription factor or sigma factor.

Two-Component System (TCS)

A bacterial signal transduction circuit consisting of a membrane-bound sensor histidine kinase and a cytoplasmic response regulator linked through a His-to-Asp phosphotransfer relay.

Sensor Histidine Kinase

A transmembrane receptor protein in a two-component system that senses environmental or periplasmic signals and autophosphorylates a conserved histidine residue using ATP.

Response Regulator

The cytoplasmic component of a two-component system that receives a phosphoryl group from a histidine kinase on an aspartate residue, activating its DNA-binding domain to alter transcription.

His-to-Asp Phosphotransfer

The defining biochemical transfer of a high-energy phosphate group from the conserved histidine of a sensor kinase to the conserved aspartate of a response regulator in bacterial signal transduction.

Alternative Sigma Factors

Specialised sigma subunits that swap out $\sigma^{70}$ in RNA polymerase to redirect the core enzyme toward completely distinct, genome-wide sets of promoters during environmental stress or physiological transitions.

Anti-Sigma Factor

A dedicated inhibitory protein (such as RseA) that binds and sequesters an alternative sigma factor (such as $\sigma^{24}$), keeping it inactive until a specific stress-induced signal triggers its degradation.

SOS Response

A coordinated bacterial global DNA damage response system regulated by RecA and LexA that derepresses DNA repair, cell division arrest, and translesion synthesis genes in an escalating temporal order.

RecA*

The active nucleoprotein filament formed when RecA binds single-stranded DNA (ssDNA) generated by DNA damage; it acts as a coprotease to stimulate the autocatalytic cleavage of LexA.

LexA

The master repressor of the bacterial SOS response that binds to SOS boxes across more than 40 promoters under normal conditions and undergoes irreversible self-cleavage upon stimulation by RecA*.

SOS Boxes

Specific operator DNA sequences bound by LexA with varying affinities, establishing a tiered temporal escalation programme where weak-affinity promoters derepress earlier than high-affinity promoters.

Intrinsic Termination

Factor-independent transcription termination mediated by the formation of an RNA hairpin from a GC-rich inverted repeat followed immediately by a run of 7–8 uracil residues (U-tract).

Rho ($\rho$) Factor

A hexameric, ring-shaped ATP-dependent RNA translocase and helicase that tracks along nascent RNA in the $5'\rightarrow 3'$ direction to unwind the RNA:DNA hybrid and release paused RNA polymerase.

rut Site (Rho Utilization Site)

An unstructured, cytosine-rich sequence of approximately 70–80 nucleotides on the nascent RNA transcript that serves as the loading platform for the Rho termination factor.

Transcriptional Read-Through

The phenomenon whereby RNA polymerase bypasses an inefficient termination site and continues transcribing downstream genes, thereby adjusting the relative expression ratio of adjacent genetic modules.

Attenuation (Trp Operon)

A regulatory mechanism linking translation speed to transcription termination, where ribosome movement across tryptophan codons in the trpL leader controls whether a terminator or antiterminator hairpin forms.

Antiterminator Hairpin (Trp Operon)

An alternate RNA stem-loop structure formed by the base pairing of regions 2 and 3 in the trpL leader transcript when the ribosome stalls at Trp codons under low tryptophan conditions, permitting continued transcription.

Riboswitch

A cis-acting regulatory segment in the $5'\text{ UTR}$ of an mRNA that directly binds a specific small-molecule ligand to modulate transcription termination or translation initiation without requiring regulatory proteins.

Aptamer Domain

The three-dimensional, highly structured RNA binding region of a riboswitch that selectively binds a specific small-molecule ligand with high affinity.

Expression Platform

The structural domain of a riboswitch that undergoes a secondary conformational shift in response to ligand binding at the aptamer, physically implementing the decision to terminate or continue transcription.

transcription initation and elongation mechanism

closed complex, RNAP binds promotor —> open complex, DNA unwinding occurs —> absortive initiation, short RNAs released —> promotor escape, prodcutive transcription begins —> elongation, RNAstrand grows

How does the bacterial RNA polymerase holoenzyme recognise promoters, and what is the role of the housekeeping sigma factor in sequence specificity?

Promoter recognition is mediated by the $\sigma$ factor associating with the core enzyme ($\alpha_2\beta\beta'\omega$) to form the holoenzyme. The housekeeping factor ($\sigma^{70}$) specifically binds the $-35$ element ($\text{TTGACA}$) and $-10$ element ($\text{TATAAT}$), separated by an optimal spacer of $\sim 17\,\text{bp}$. Base variations at critical positions (especially within the $-10$ motif) significantly alter binding affinity and basal transcription levels.

What distinct catalytic roles do the $\beta$ and $\beta'$ subunits of bacterial RNA polymerase perform during transcription?

Together they form the catalytic core and 'crab claw' structure: the $\beta$ subunit catalyses nucleotide addition onto the growing RNA transcript, while the $\beta'$ subunit coordinates catalytic $\text{Mg}^{2+}$ ions required for phosphodiester bond chemistry.

What structural and regulatory functions do the $\alpha$ and $\omega$ subunits serve in bacterial RNA polymerase?

Two $\alpha$ subunits act as scaffolds for holoenzyme assembly, with their flexible C-terminal domains ($\alpha\text{CTD}$) contacting upstream DNA UP elements and activator proteins to recruit polymerase. The $\omega$ subunit functions as an assembly chaperone that helps the $\beta'$ subunit fold properly.

What molecular events define closed complex formation during bacterial transcription initiation?

The RNA polymerase holoenzyme sequence-specifically binds to the promoter while the target DNA duplex remains completely double-stranded. This initial binding intermediate is further stabilized when the $\alpha\text{CTD}$ interacts with upstream UP elements.

What conformational change occurs during open complex formation (isomerization) in transcription initiation?

The DNA duplex is locally melted, unwinding approximately $13\,\text{bp}$ around the transcription start site to create the transcription bubble. This exposes a short stretch of single-stranded template DNA to the RNA polymerase active site.

Why does RNA polymerase undergo abortive initiation before progressing to processive elongation?

During early transcription initiation, RNA polymerase repeatedly synthesises short transcripts of a few nucleotides and releases them without escaping the promoter, because the enzyme remains constrained by promoter contacts and the bound $\sigma$ factor.

What structural transition enables RNA polymerase to achieve promoter escape (clearance)?

Once RNA polymerase synthesises a transcript exceeding approximately $10\,\text{nt}$, it undergoes a conformational change that prompts the release of the $\sigma$ factor, breaking promoter contacts and moving into elongation.

How does RNA polymerase maintain processivity and separate nascent RNA during transcription elongation?

The enzyme travels along the template DNA maintaining a $13\,\text{bp}$ transcription bubble; internal rudder and lid domains physically peel the newly synthesised RNA away from the DNA template, allowing the DNA duplex behind the bubble to re-anneal.

What is the five-step sequential mechanism of bacterial transcription initiation and elongation?

1. Closed complex formation: holoenzyme binds double-stranded promoter DNA. 2. Open complex formation (isomerization): roughly $13\,\text{bp}$ melts to form the transcription bubble. 3. Abortive initiation: short transcripts (<$10\,\text{nt}$) are repeatedly synthesised and released. 4. Promoter escape: transcript exceeds $10\,\text{nt}$, triggering a conformational change and $\sigma$ release. 5. Elongation: enzyme moves along DNA, using rudder and lid domains to peel RNA off template DNA while the duplex re-anneals.

What structural architecture enables bacterial transcription factors to bind specific operator sequences?

Most bacterial transcription factors are homodimers that recognise partially symmetrical operator sequences using a conserved helix-turn-helix motif, in which one recognition $\alpha$-helix makes sequence-specific contacts in the major groove of DNA.

By what two spatial mechanisms can repressor proteins inhibit bacterial transcription?

1. Steric hindrance: the repressor binds an operator directly overlapping promoter motifs (e.g. $-10$ or $-35$), physically excluding RNA polymerase. 2. DNA looping: a multimeric repressor simultaneously binds two non-contiguous operators, looping the intervening DNA to block polymerase access.

How do bacterial transcriptional activators stimulate transcription from weak promoters?

Activators bind specific target operators upstream of the promoter and make direct protein-protein contacts with the $\alpha\text{CTD}$ of RNA polymerase, recruiting and stabilising the holoenzyme at the promoter to increase the rate of closed complex formation.

How does the LacI repressor physically block transcription of the lac operon?

LacI functions as a homotetramer formed from two dimers that binds simultaneously to the proximal operator and one of two distal operators, looping the intervening DNA across the promoter to provide stable transcriptional repression.

What biochemical mechanism allows allolactose to relieve LacI-mediated repression of the lac operon?

Allolactose, a metabolic byproduct produced by basal $\beta$-galactosidase activity, binds to the regulatory domain of LacI. This induces an allosteric conformational change that reduces its affinity for operator DNA, causing LacI to dissociate.

Why is leaky promoter expression essential for the induction of bacterial operons like lac?

Because repression is never completely absolute, low basal transcription produces small baseline quantities of permease (LacY) and $\beta$-galactosidase (LacZ). These basal proteins are required to transport initial lactose into the cell and isomerise it into the allolactose inducer.

What distinct functional advantages do negative and positive autoregulatory gene circuits provide?

In negative autoregulation, a transcription factor represses its own promoter, accelerating response times and stabilising protein concentrations against fluctuations. In positive autoregulation, a factor activates its own promoter, creating positive feedback that can establish bistability (two stable, persistent expression states).

How does glucose availability regulate intracellular cyclic AMP (cAMP) levels in bacteria?

Glucose transport through the phosphotransferase system (PTS) inhibits adenylate cyclase. When glucose is abundant, adenylate cyclase remains inhibited, keeping cAMP low; when glucose is depleted, adenylate cyclase is active, synthesising high levels of cAMP from ATP.

How does the cAMP receptor protein (CRP) activate transcription upon glucose starvation?

Elevated intracellular cAMP binds CRP (CAP), inducing an allosteric conformational shift that allows CRP to bind target promoter DNA. The bound CRP dimer then recruits RNA polymerase to the promoter via direct contacts with the polymerase $\alpha\text{CTD}$.

How do Class I and Class II CRP-activated promoters differ in their architecture and polymerase contacts?

In Class I promoters (such as $P_{\text{lac}}$), CRP binds upstream of core promoter elements and contacts only the $\alpha\text{CTD}$. In Class II promoters, CRP binds overlapping the $-35$ region and directly contacts both the $\alpha\text{CTD}$ and the $\sigma$ subunit of RNA polymerase.

Why does the lac operon behave as a molecular AND-gate, achieving maximal expression under only one specific environmental condition?

Maximal transcription requires two simultaneous inputs: lactose must be present to form allolactose and relieve LacI repression, and glucose must be absent so that high cAMP-CRP can actively recruit RNA polymerase to the promoter.

What biochemical sequence of events transduces environmental signals across a bacterial two-component system (TCS)?

A transmembrane sensor histidine kinase detects an environmental stimulus and autophosphorylates a conserved histidine residue using ATP. It then carries out a His-to-Asp phosphotransfer to a conserved aspartate residue on a partner response regulator, activating its DNA-binding domain to alter target gene transcription.

By what mechanisms do bacterial cells control the activity of alternative sigma factors competing for limited core enzyme?

Because core RNA polymerase is limiting, alternative sigma factors compete for binding. Their activity is regulated via stoichiometric sequestration by dedicated anti-sigma factors (e.g. RseA holding $\sigma^{24}$ inactive until stress triggers its proteolysis) or by regulated protein stability (e.g. $\sigma^{38}$ undergoing rapid degradation under non-stress conditions).

What molecular events trigger derepression of the SOS regulon following bacterial DNA damage?

DNA damage produces single-stranded DNA (ssDNA), which is bound by RecA to form active $\text{RecA}^*$ nucleoprotein filaments. $\text{RecA}^*$ acts as a coprotease that stimulates the master repressor LexA to undergo irreversible autocatalytic self-cleavage, relieving repression across SOS promoters.

Why does the bacterial SOS response follow a tiered temporal escalation programme rather than derepressing all genes at once?

SOS boxes have different binding affinities for LexA. Promoters with weak-affinity operators lose repression early at low damage to express accurate excision repair and cell division arrest, whereas promoters with high-affinity operators derepress only under extensive damage to activate mutagenic, error-prone translesion synthesis as a last resort.

What two RNA sequence elements mediate intrinsic (factor-independent) termination, and what are their physical roles?

A GC-rich inverted repeat transcribes into an RNA hairpin that jams into the polymerase RNA exit channel, mechanically pausing and destabilizing the elongation complex. An immediately adjacent downstream U-tract forms an exceptionally weak rU:dA hybrid with the template DNA, causing the paused complex to dissociate.

How does the Rho factor find, chase, and dissociate a transcribing RNA polymerase elongation complex?

Rho binds an unstructured, cytosine-rich rut site ($\sim 70\text{--}80\,\text{nt}$) on nascent RNA and translocates in the $5'\rightarrow 3'$ direction via ATP hydrolysis. When RNA polymerase pauses at specific pause sites, Rho catches up and uses its helicase activity to unwind the RNA:DNA hybrid, releasing polymerase, RNA, and DNA.

How do variations in termination efficiency allow bacteria to regulate the expression ratios of adjacent genes (transcriptional read-through)?

An inefficient terminator allows a defined fraction of transcribing RNA polymerases to bypass the termination signal and continue transcribing downstream genes, establishing a fixed stoichiometric ratio of mRNA and protein expression between adjacent genetic modules.

How does transcription-coupled translation regulate the trp operon through attenuation under low versus high tryptophan conditions?

Under high tryptophan, ribosomes translate rapidly across two leader Trp codons in trpL, allowing regions 3 and 4 to pair into an intrinsic terminator hairpin that halts transcription. Under low tryptophan, ribosomes stall at the Trp codons, allowing regions 2 and 3 to pair into an antiterminator hairpin, which prevents terminator formation and permits complete transcription of biosynthesis genes.

How do riboswitches regulate transcription termination in the absence of regulatory proteins?

Located in the $5'\text{ UTR}$, a riboswitch consists of an aptamer domain that binds a specific small-molecule metabolite and an adjacent expression platform. When the metabolite binds the aptamer, it stabilizes a conformation that sequesters antiterminator sequences, forcing the expression platform into an intrinsic terminator hairpin that prematurely stops transcription.