Regulation of Gene Expression in Prokaryotes

What is an operon in prokaryotes?

An operon is a cluster of coordinately regulated genes under the control of a single promoter and operator. It includes structural genes, regulatory genes, and regulatory sites (promoters/operators) that control transcription as one unit.

What are the components of an operon?

1. Structural genes – code for proteins or enzymes. 2. Regulatory genes – encode repressors or activators. 3. Regulatory sites – promoters and operators that control RNA polymerase binding and gene transcription.

What is a cistron?

A cistron is a single coding sequence or open reading frame (ORF) within an operon that encodes one protein.

What is a polycistronic mRNA?

An mRNA that contains multiple ORFs (coding sequences) derived from a single promoter—common in prokaryotes. Each cistron encodes a separate protein.

What sequence precedes the start codon in prokaryotic mRNA to help ribosome binding?

The Shine-Dalgarno box, located upstream of the start codon (AUG), aligns the ribosome with the start site for translation initiation.

What are the -35 and -10 promoter sequences?

Consensus DNA sequences recognized by RNA polymerase sigma factor. The -35 region (TTGACA) and -10 region (TATAAT) determine promoter strength and transcription efficiency.

How do repressors regulate transcription?

Repressors bind to the operator region and block RNA polymerase access to the promoter, preventing transcription.

How do activators regulate transcription?

Activators bind to regulatory sites near promoters, enhancing RNA polymerase binding and transcription initiation (increase KB).

Differentiate positive and negative control.

Positive control: a regulatory protein (activator) is required for transcription (operon ON). Negative control: a regulatory protein (repressor) binds to inhibit transcription (operon OFF).

What defines inducible vs. repressible operons?

Inducible operons are turned ON in the presence of a metabolite (e.g., lac operon with lactose). Repressible operons are turned OFF in the presence of a metabolite (e.g., trp operon with tryptophan).

What are the genes in the lac operon?

Regulatory gene (lacI), control sites (promoter P, operator O), and structural genes (lacZ, lacY, lacA) encoding β-galactosidase, lactose permease, and β-galactoside transacetylase.

What enzyme does lacZ code for, and what is its function?

LacZ encodes β-galactosidase, which breaks lactose into glucose and galactose.

When is the lac operon repressed?

In the absence of lactose, the lac repressor binds to the operator, blocking RNA polymerase and preventing transcription.

When is the lac operon induced (derepressed)?

In the presence of lactose (allolactose), the inducer binds the repressor, releasing it from the operator, allowing transcription of lacZYA.

What role does allolactose play in the lac operon?

Allolactose is the natural inducer that binds the lac repressor, causing it to detach from the operator and enabling transcription.

What is the role of cAMP and CAP in the lac operon?

In low glucose conditions, cAMP levels rise and bind to CAP (catabolite activator protein). The cAMP-CAP complex binds upstream of the promoter, enhancing RNA polymerase binding and transcription.

What happens to cAMP levels in presence of glucose?

cAMP levels decrease, CAP cannot bind DNA, and lac operon transcription remains low even if lactose is present (catabolite repression).

What are the conditions for maximum lac operon transcription?

High cAMP (low glucose) and presence of lactose (inducer). This ensures both activation by CAP and derepression by allolactose.

What enzymes are produced by the lac operon when expressed?

β-galactosidase (LacZ), lactose permease (LacY), and β-galactoside transacetylase (LacA).

What is the function of the trp operon?

It encodes enzymes for tryptophan biosynthesis. When tryptophan is abundant, transcription is repressed or attenuated.

How does the trp repressor regulate transcription?

In the absence of tryptophan, the aporepressor is inactive and cannot bind the operator (transcription ON). In the presence of tryptophan, it binds the aporepressor to form the active repressor complex, which blocks transcription (OFF).

What is attenuation in the trp operon?

A second regulatory mechanism where transcription termination depends on the formation of stem-loop structures in the leader mRNA, controlled by [trp-tRNA].

What determines whether transcription continues or stops during attenuation?

High tryptophan allows the ribosome to translate quickly, forming a 3-4 terminator stem-loop → transcription stops. Low tryptophan causes ribosome stalling, forming a 2-3 anti-terminator loop → transcription continues.

What is the leader peptide in the trp operon?

A short 14-amino-acid sequence in the leader region, containing two tryptophan codons that sense intracellular tryptophan levels.

What structure forms during high tryptophan conditions?

A 3-4 stem-loop structure (terminator) forms, causing premature termination of transcription.

What structure forms during low tryptophan conditions?

A 2-3 stem-loop (anti-terminator) forms, allowing RNA polymerase to transcribe the structural genes (trpE–trpA).

Summarize attenuation in one sentence.

Attenuation is a mechanism that fine-tunes trp operon transcription based on tryptophan availability by altering leader mRNA secondary structure.

List the three main mechanisms of transcription activation in prokaryotes.

1. Regulated Recruitment (e.g., CAP recruits RNAP). 2. RNA Polymerase Activation (e.g., NtrC with σ⁵⁴). 3. Promoter Activation (e.g., MerR twisting DNA or bending by HU/IHF/FIS).

Explain regulated recruitment.

An activator such as CAP interacts with the α-subunit (α-CTD) of RNA polymerase, facilitating its recruitment to the promoter.

Explain RNA polymerase activation (NtrC example).

NtrC hydrolyzes ATP to activate σ⁵⁴-containing RNA polymerase, enabling it to unwind DNA and begin transcription.

Explain promoter activation by DNA twist (MerR example).

MerR binds the merT promoter and, upon binding mercury, twists DNA to align -10 and -35 promoter elements correctly, allowing transcription.

What role do DNA-bending proteins play in transcription?

Proteins like HU, IHF, and FIS bend DNA, helping activators and RNA polymerase interact more efficiently to initiate transcription.

What happens in a mutation of the lac operator?

Repressor cannot bind; operon is constitutively expressed (always ON).

What happens in a mutation of the lacI gene?

No repressor produced; lac operon is always ON (constitutive expression).

What happens in a mutation of the lac promoter?

RNA polymerase cannot bind efficiently; lac operon is OFF regardless of inducer.

What determines ribosome progress in trp attenuation?

The availability of charged tryptophanyl-tRNA; it determines whether translation pauses at the trp codons in the leader peptide.