RA

Regulation of Gene Expression in Bacteria (Lectures 13-14)

Promoter Architecture and Transcription Initiation

  • Transcription initiation in many E. coli promoters relies on two key promoter elements bound by RNA polymerase: the -35 element and the -10 element.
    • -35 element sequence often listed as TTGACA.
    • -10 element sequence often listed as TATAAT.
    • The region between these elements and the transcription start site includes spacer sequences (e.g., N17 between -35 and -10, or variations like N7, N19).
  • Strong promoters have sequences that bind RNA polymerase well; examples mentioned:
    • recA promoter (strong)
    • araBAD promoter (weak)
  • The exact promoter strength arises from both the specific base sequence and the spacer length between the -35 and -10 elements.
  • Important note references:
    • Einav & Phillips, 2019, PNAS 116:13340-13345, on optimal spacing between -35 and -10 elements (distance matters more than exact sequence for transcription initiation).
  • Optimal spacing concept:
    • RNA polymerase contacts both the -35 and -10 regions when spacing is correct.
    • If spacing is too long or too short, RNA polymerase cannot contact both regions effectively, reducing transcription efficiency.
  • Readings and foundational references provided include Griffiths et al. and Pierce texts for deeper coverage on promoter architecture.

Information Flow and Regulation (General Model)

  • Information flow through the central dogma and its regulation:
    • DNA → transcription → RNA → translation → protein folding → activity/function
  • Variability in gene expression arises from differences in gene copy number, transcription efficiency, RNA stability, translation efficiency, ribosome binding, codon usage, protein stability, and post-translational modifications.
  • Gene copy number can vary widely: plasmids can exist in tens to hundreds of copies per cell, impacting overall product levels.
  • Post-transcriptional and post-translational controls can substantially alter final protein activity even with similar transcription levels.
  • A schematic evolution of control points:
    • Transcription efficiency
    • mRNA stability (half-life; bacterial mRNA half-life ~ t_{1/2} \approx 2 \,\text{min})
    • Translation initiation (ribosome binding efficiency)
    • Translation rate (codon usage, tRNA abundance)
    • Protein stability
    • Post-translational modifications and folding
  • These factors collectively determine final protein activity and cellular phenotype.

The Lac Operon: Structure, Regulation, and Mutations

  • Components:
    • lacI (lac repressor)
    • lacZ (β-galactosidase)
    • lacY (lactose permease)
    • lacA (transacetylase)
  • Regulation is primarily a negative control system with an inducible response:
    • In the absence of lactose, the lac repressor binds to the operator and blocks transcription.
    • In the presence of lactose, the inducer is actually allolactose (the effector molecule) which binds the lac repressor, causing a conformational change that reduces operator binding, allowing transcription.
  • Mutations and their classic phenotypes:
    • Oc mutations: constitutive operators that prevent repressor binding to the operator; the operon is always on (cis-acting effect).
    • lacI- mutations: defective repressor protein that cannot bind the operator, so operon is always on (trans-acting effect when a wild-type lacI is present elsewhere in the cell).
    • lacI+ gene product (repressor) can act in trans to repress lac operons, even if another lac operon is mutated, illustrating trans-acting regulation.
    • I+ vs I- designations illustrate whether the repressor is functional (I+ can repress; I- cannot).
  • Cis vs trans considerations:
    • Cis-acting elements (e.g., Oc) affect only the nearby operon on the same DNA molecule.
    • Trans-acting factors (e.g., LacI repressor) can act on multiple DNA sites across the cell, including other operons, provided the repressor is functional.
  • Catabolite repression of the lac operon (global control):
    • Expression of lactose-metabolizing enzymes requires two conditions:
    • Lactose must be present (negative regulation is relieved by allolactose).
    • Glucose must be absent (positive regulation via cAMP and CAP).
    • Mechanism:
    • Glucose levels inversely regulate intracellular cAMP levels: more glucose → lower cAMP; less glucose → higher cAMP.
    • cAMP binds CAP (catabolite activator protein); the CAP-cAMP complex enhances transcription by interacting with RNA polymerase to improve binding to the -10/-35 promoter regions.
  • Lac operon control logic recap:
    • When glucose is present: low cAMP → CAP activation is reduced → transcription is least favored.
    • When lactose is present: allolactose binds the repressor, lifting repression.
    • The full expression of lac genes typically requires lactose presence and glucose absence (logical AND of de-repression and CAP activation).
  • Key dynamic observations:
    • Jacob & Monod observed that in the presence of lactose, E. coli can produce up to ~1000x more β-galactosidase; kinetics can be rapid (e.g., production observed in ~3 minutes) illustrating an efficient on/off switch in response to environmental cues.
  • Regulatory circuit logic and mutations in lac components can be predicted to yield various phenotypes (on/off, constitutive expression, etc.), enabling exploration of cis/trans effects and promoter strength.

The Arabinose Operon: Dual Positive and Negative Control

  • The ara operon encodes the activator AraC, which can mediate both positive and negative regulation depending on arabinose availability.
  • Dual positive control in the presence of arabinose:
    • AraC binds to araI to initiate transcription (positive control).
    • CAP-cAMP system also provides positive control, reinforcing transcription when arabinose is present.
  • Negative control in the absence of arabinose:
    • AraC binds to araO and araI, forming a DNA loop that prevents RNA polymerase access to the promoter, thereby repressing transcription.
  • AraC binding sites and conformational changes:
    • In the presence of arabinose, AraC undergoes a conformational change favoring binding to two araI sites, promoting transcription.
    • In the absence of arabinose, AraC forms a dimmer that binds araO and araI to repress transcription.
  • Two AraC binding site arrangements:
    • Two AraC binding sites at araI enable AraC to function as an activator under inducing conditions when arabinose is present.
    • DNA looping mechanism ensures tight repression when arabinose is absent.
  • Conceptual takeaway:
    • The arabinose operon exemplifies a regulatory switch that can simultaneously integrate multiple signals (arabinsose availability and CAP-cAMP status) to finely tune gene expression.
  • Key reference:
    • Schleif, 2010, FEMS Microbiology Reviews, 34:779–96, on AraC conformations and binding dynamics.

Global Gene Regulation and Sigma Factors

  • Global regulation coordinates broad gene expression programs via sigma factors and promoter recognition.
  • Transcriptional control basics:
    • Promoter defines where transcription starts and how strongly RNAP binds.
    • Sigma factors are subunits of RNA polymerase that confer promoter specificity.
  • Sigma factors:
    • Sigma factor is a detachable RNA polymerase subunit that recognizes promoter DNA sequences.
    • When associated with RNAP, sigma factors bind specific DNA motifs in promoters to initiate transcription; after transcription starts, the sigma factor is released and recycled.
  • Canonical promoter recognition:
    • In E. coli, sigma factor 70 recognizes the canonical -35 and -10 promoter sequences (-35: TTGACA; -10: TATAAT) with appropriate spacer and context.
  • Diversity of sigma factors:
    • Most bacteria possess multiple sigma factors that redirect RNA polymerase to different subsets of genes, enabling coordinated responses (e.g., heat shock, stationary phase, sporulation, stress responses).
    • A single sigma factor can regulate large suites of genes (orange promoters in the schematic), while alternative sigma factors regulate pathway-specific genes (blue promoters).
  • Conceptual implication:
    • Global reprogramming of gene expression can be achieved by altering the expression or activity of sigma factors, enabling broad, coordinated shifts in cellular physiology.

Consensus Sequences and Promoter Recognition

  • Definition:
    • A consensus sequence summarizes the most commonly observed nucleotides at specific positions across multiple aligned sequences (DNA, RNA, or protein sites).
    • Consensus sequences are generated by multiple sequence alignments and describe DNA-binding sites and promoter motifs.
  • Majority-rule consensus:
    • The most frequent nucleotide at each position defines the consensus.
    • Example for the -35 box: TNATATT (note that some positions have no strict consensus, hence degenerate letters).
  • -10 box consensus:
    • The consensus for the -10 box is typically closer to TATAAT, though individual promoters show variation; the exact consensus is inferred from alignments and may include degenerate positions.
  • Practical implications:
    • The closer a binding site matches the consensus, the stronger the likely binding and transcription initiation.
    • However, actual promoter strength also depends on spacing, context, and regulatory proteins.
  • Visual reference and examples:
    • The -35 box and -10 box sequences are common across many E. coli promoters; examples include recA (strong) and araBAD (weak).
  • Additional readings and definitions:
    • The concept of consensus sequences is widely used for describing transcription factor binding sites and is illustrated in Nature Scitable definitions.

Genetic Switches, Cis/Trans Regulation, and Logic Circuits

  • Regulatory proteins controlling transcription:
    • Promoter determines where transcription begins.
    • Operator is the binding site for repressors.
    • Absence of repressor allows transcription; activator binding site is another site for activator proteins.
  • Activators and repressors respond to environmental cues, enabling logic-like control of gene expression.
  • Two key functional sites in activator/repressor proteins:
    • DNA-binding site: allows interaction with operator/promoter DNA.
    • Allosteric site: binds effector molecules to modulate DNA-binding conformation.
  • Genetic logic circuits:
    • By combining activators and repressors that respond to different signals (e.g., lactose, glucose, arabinose), cells implement logical operations (AND, OR, NOT) at the transcriptional level.
    • For example, lac operon logic uses a repressor responsive to lactose (present/absent) and an activator (CAP-cAMP) responsive to glucose levels.
  • Design exercises and thought experiments:
    • Propose a circuit where operon expression occurs when serine is lacking and pyruvate is present, and add a condition for expression only when oxygen is present.
    • Explore how adding separate repressors for each input could implement alternative logic circuits.
  • Complexity with increasing gene numbers:
    • As more genes are regulated, the number of regulatory genes increases disproportionately due to combinatorial control requirements.
    • Visualized concept: red regulatory genes, other colored genes represent different functional categories (Van Nimwegen 2003).

Post-Transcriptional and Post-Translational Regulation

  • Post-transcriptional controls include:
    • mRNA stability and half-life, mRNA modifications, and processing.
    • Translation efficiency, influenced by ribosome binding, initiation rate, and codon usage.
  • Post-translation controls include:
    • Protein folding, maturation, stability, and post-translational modifications.
    • Protein activity can be modulated independently of transcription levels.
  • The broad regulatory picture: regulation acts at multiple levels to fine-tune the final protein activity in response to cellular conditions.

Practical and Ethical/Philosophical Implications

  • Understanding gene regulation in bacteria informs broader concepts of systems biology, synthetic biology, and metabolic engineering.
  • Engineering genetic circuits touches on ethics of biosafety, unintended ecological impact, and responsible innovation.
  • The balance between natural regulatory complexity and synthetic simplification is a key consideration in practical applications.

Learning Objectives (Summary)

  • Describe how lactose and arabinose operons are regulated and why their regulation is organized this way.
  • Explain how to predict phenotypes of mutations in components of genetic control systems for these operons.
  • Design a novel genetic control circuit to achieve a desired expression outcome using activators and/or repressors.
  • Explain how sigma factors enable large-scale reprogramming of gene expression networks.
  • Define and determine a majority-rule consensus sequence from a multiple sequence alignment for promoter elements.
  • Understand promoter architecture, including the -35 and -10 elements, spacer requirements, and spacing effects on transcription initiation.
  • Distinguish cis-acting elements from trans-acting factors and understand their genetic consequences.
  • Recognize the concept of catabolite repression and how CAP-cAMP integrates metabolic state with transcriptional control.
  • Appreciate the global regulatory architecture that enables bacteria to coordinate gene expression across large sets of genes through sigma factors and promoter recognition.

References from Transcript Pack

  • Griffiths et al. (12th ed) 11.1-11.4, 11.7; Griffiths et al. (11th ed) 11, p381-413; Pierce (6th ed) Chap 16, pp. 461-482.
  • Einav & Phillips (2019) PNAS 116:13340-13345 on promoter spacing and binding dynamics.
  • Schleif (2010) FEMS Microbiology Reviews 34:779–96 on AraC architecture and arabinose operon regulation.
  • Jacob & Monod classic experiments on lac operon regulation and mutational analysis.
  • General definitions: consensus sequence from Nature Scitable and related genome regulation resources.

Quick recap of key concepts

  • Lac operon: glucose absence and lactose presence create an AND-like logic via CAP-cAMP activation and lac repressor release, respectively.
  • Arabinose operon: dual positive and negative control via AraC and CAP-cAMP; arabinose presence shifts AraC to promote transcription, absence promotes looping and repression.
  • Promoter architecture: -35 and -10 elements with spacer determine transcription initiation efficiency; spacing is critical for RNAP contact.
  • Sigma factors: enable global reprogramming by switching promoter recognition; multiple sigma factors coordinate different cellular responses.
  • Cis vs trans: cis elements affect nearby genes; trans factors can act genome-wide.
  • Consensus sequences: provide a probabilistic description of promoter motifs; the closer to the consensus, the stronger the predicted binding.
  • Regulation is multi-layered: transcriptional control is complemented by post-transcriptional, translational, and post-translational mechanisms to fine-tune gene expression and phenotype.