Prokaryotic Gene Expression II

Why is gene regulation required in prokaryotes?

To conserve energy and resources; rapidly respond to environmental changes; coordinate expression for specific functions; and maintain cellular homeostasis.

What are constitutively active genes?

Genes expressed under a broad range of conditions.

What are housekeeping genes, and what are examples given in the notes?

Constitutively active genes involved in essential functions, such as ribosome function and core metabolism.

What characterises regulated genes?

They are expressed in response to environmental signals, can be activated or repressed, and are controlled by regulatory proteins or small molecules.

Which levels of gene expression can be regulated? (Multiple answer) A. Transcriptional B. Translational C. Post-translational D. DNA replication

A, B and C.

What are three mechanisms or systems through which gene regulation occurs?

Regulatory proteins, regulatory RNA, and global regulation, such as through sigma factors and second messengers.

What do regulatory proteins bind to, and what do they control?

They bind to specific DNA sequences and control the initiation of transcription.

What is the function of a repressor protein?

To prevent transcription.

What is the function of an activator protein?

To activate transcription.

How can ligand binding affect regulatory proteins?

Regulatory proteins are often allosterically modified by ligand binding; the ligand is usually a substrate or product of the regulated pathway.

How can proteins recognise specific sequences in double-stranded DNA?

Base-pair edges are exposed, and each base-pair orientation presents a unique chemical pattern that proteins can recognise.

Which DNA groove do regulatory proteins mainly bind?

The major groove.

What is the helix-turn-helix (HTH) motif?

A DNA-binding motif in which a recognition helix contacts the edges of base pairs through its amino acid side chains.

What are the two features of the HTH motif identified in the notes?

A recognition helix and amino acid side chains that contact base-pair edges.

What other protein–DNA contact motif is mentioned?

Ribbon-helix-helix.

Define negative gene regulation.

A repressor binds and blocks transcription.

Define positive gene regulation.

An activator binds and enhances transcription.

Define inducible gene regulation.

The system is normally off and is turned on by an inducer.

Define repressible gene regulation.

The system is normally on and is turned off by a co-repressor.

(Multiple choice) Which regulation type is normally off and switched on by an inducer? A. Positive B. Negative C. Inducible D. Repressible

C. Inducible.

(Multiple choice) Which regulation type is normally on and switched off by a co-repressor? A. Inducible B. Repressible C. Positive D. Constitutive

B. Repressible.

What is a promoter in an operon?

The site where RNA polymerase (RNAP) binds.

What is an operator in an operon?

A regulatory region.

What are structural genes?

Genes that encode proteins; they contain the coding sequences.

What are regulatory genes, and where are they located according to the notes?

Genes located elsewhere that encode repressor proteins.

What carbon source does E. coli preferentially use, and what does it use when glucose is depleted?

It preferentially uses glucose and turns to lactose when glucose is depleted.

What type of gene regulation does the lac operon use?

Negative and inducible regulation.

Why is the lac operon an example of catabolic gene regulation?

It is switched off until required for lactose utilisation.

What does lacZ encode, and what is its function?

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

What does lacY encode, and what is its function?

Lactose permease, which imports lactose into the cell.

What does lacA encode, and what is its function?

Thiogalactoside transacetylase, involved in detoxification.

What does lacI encode?

The lac repressor.

Which lac operon genes encode the proteins involved in lactose utilisation and detoxification?

lacZ, lacY and lacA.

What is the molecular mass and structure of the lac repressor?

It is a 38 kDa protein that forms a tetramer.

How does the lac repressor control the lac structural genes?

It binds to the lac operator DNA sequence and blocks transcription of lacZ, lacY and lacA.

What is the main operator site associated with lac repressor binding according to the notes?

The main operator site at the +1 site.

Which DNA-binding motif is identified for the lac repressor?

The helix-turn-helix (HTH) motif.

Why can the lac repressor form DNA loops?

Binding to auxiliary operator sites causes looping.

What is the effect of operator looping on lac operon repression?

It increases repression.

(Multiple answer) Which statements about the lac repressor are supported by the notes? A. It is a 38 kDa protein. B. It forms a tetramer. C. It binds the lac operator. D. It activates lacZ, lacY and lacA transcription.

A, B and C.

What is basal expression of the lac operon?

A constant low level of expression that produces low β-galactosidase activity.

What is allolactose?

A disaccharide similar to lactose that acts as the inducer of the lac operon.

How is allolactose produced according to the notes?

β-galactosidase occasionally performs a trans-glycosylation reaction.

Why does the lac operon not respond as soon as lactose is present?

It responds at high lactose concentrations, when enough allolactose has been produced by the occasional side reaction.

How does allolactose induce lac operon expression?

It binds allosterically to the lac repressor, induces a conformational change, and prevents the repressor from binding to the operator.

(Multiple choice) What happens when allolactose binds to the lac repressor? A. The repressor binds the operator more strongly. B. The repressor changes conformation and cannot bind the operator. C. RNA polymerase is destroyed. D. Lactose permease is inhibited directly.

B. The repressor changes conformation and cannot bind the operator.

What is IPTG?

An allolactose analogue that is stable and non-hydrolysable.

What is IPTG used for in molecular biology?

To induce the LacI-regulated system, for example during protein expression.

How does glucose availability provide another level of lac operon regulation?

The lac repressor provides negative regulation, while catabolite activator protein (CAP) provides positive regulation.

What is the function of catabolite activator protein (CAP)?

CAP is an activator protein that mediates positive control of transcription.

How does cAMP activate CAP?

cAMP binds CAP, induces a conformational change that allows CAP to bind its DNA site, and enhances RNAP binding to the promoter.

How is glucose linked to cAMP levels?

Glucose presence indirectly affects adenyl cyclase activity, which produces cAMP; in the absence of glucose, cAMP levels rise.

What happens to CAP activity when glucose is present?

cAMP levels are low, so CAP is inactive.

What happens to cAMP and CAP when glucose is absent?

cAMP is synthesised and its binding activates CAP, allowing CAP to bind DNA and enhance RNAP binding to the promoter.

What is the CAP–cAMP regulon?

A system in which CAP sites occur at hundreds of promoters and coordinate cellular responses to carbon availability.

(Multiple answer) Which statements about CAP–cAMP regulation are correct? A. CAP mediates positive regulation. B. cAMP binds CAP. C. In the absence of glucose, cAMP levels rise. D. CAP sites occur at hundreds of promoters. E. Glucose absence always inactivates CAP.

A, B, C and D.

How does the lac operon integrate negative and positive regulation?

The lac repressor provides negative control associated with lactose induction, while CAP provides positive control linked to glucose availability through cAMP.

What type of regulation does the trp operon use?

Negative, repressible regulation.

What is the function of the trp operon?

It includes genes required to synthesise tryptophan.

How does the trp operon respond to tryptophan levels?

It responds to tryptophan concentration and is an example of anabolic regulation that remains on until tryptophan is no longer required.

Why is the trp operon described as repressible?

Tryptophan acts as a co-repressor, allowing the repressor to bind the operator and turn transcription off.

How many tryptophan molecules bind to the trp repressor according to the notes?

Two tryptophan molecules bind to the homodimer repressor.

What is the effect of tryptophan binding to the trp repressor?

It induces a conformational change that allows the repressor to bind the operator.

What is the structure of the trpR repressor?

It is a homodimer.

How does tryptophan affect the DNA-binding ability of the trp repressor?

Tryptophan stabilises the high-affinity DNA-binding conformation.

Which DNA-binding motif is identified in the trp repressor?

The helix-turn-helix (HTH) motif.

Besides repressor binding, what other factor controls transcription of the trp operon?

The abundance of charged (loaded) tRNA^Trp.

Why does trp operon attenuation rely on transcription–translation coupling?

Ribosome movement along the leader sequence affects which mRNA secondary structure forms, thereby influencing whether transcription continues or terminates.

What is attenuation in the trp operon?

A second mechanism that fine-tunes transcription through formation of alternative mRNA hairpin structures in the leader sequence.

What is encoded by the 5′ leader sequence of the trp operon?

A 14-amino-acid leader peptide containing two consecutive tryptophan residues.

How many regions are involved in the trp operon leader mRNA, and what secondary structures can they form?

There are four regions that can form hairpin secondary structures; region 3 is complementary to regions 2 and 4.

What effect can hairpin structures have on RNA polymerase during trp operon attenuation?

They can pause RNA polymerase.

What happens during trp operon attenuation when tryptophan levels are low?

Charged tRNA^Trp is limited, so the ribosome pauses at the pair of tryptophan codons in region 1 while waiting for tRNA^Trp.

What happens to the leader mRNA when tryptophan levels are low?

Regions 2 and 3 form an anti-terminator loop, allowing transcription to continue.

What happens during trp operon attenuation when tryptophan levels are high?

Charged tRNA^Trp is abundant, so the ribosome moves quickly through region 1 without waiting.

What happens to the leader mRNA when tryptophan levels are high?

Regions 3 and 4 form a terminator loop, a hairpin followed by a poly-U sequence, and transcription is aborted.

(Multiple choice) Under low-tryptophan conditions, which structure forms in the trp leader mRNA? A. Region 3–4 terminator loop B. Region 2–3 anti-terminator loop C. A loop that always aborts transcription D. No secondary structure

B. Region 2–3 anti-terminator loop.

(Multiple choice) Under high-tryptophan conditions, what causes transcription of the trp operon to be aborted? A. The ribosome pauses at region 1. B. Regions 2 and 3 form an anti-terminator. C. Regions 3 and 4 form a terminator hairpin followed by poly-U. D. Charged tRNA^Trp becomes limited.

C. Regions 3 and 4 form a terminator hairpin followed by poly-U.

(Multiple answer) Which features are associated with the trp leader sequence? A. A 14-amino-acid leader peptide. B. Two consecutive Trp residues. C. Four mRNA regions. D. Region 3 complementary to regions 2 and 4. E. Attenuation occurs without transcription–translation coupling.

A, B, C and D.

Compare the roles of repression and attenuation in trp operon regulation.

The repressor responds directly to tryptophan concentration, while attenuation fine-tunes transcription according to charged tRNA^Trp abundance through transcription–translation coupling and alternative leader mRNA structures.

What is the basic mechanism of RNA-based gene regulation?

It relies on base pairing and the formation of RNA secondary structures or complexes between RNA molecules.

What are three possible outcomes of RNA-based gene regulation?

Transcription termination, translation control, and changes in mRNA stability or degradation.

How can RNA folding regulate gene expression?

Regulatory elements within an mRNA transcript fold into structures that control expression; examples include attenuation, riboswitches and thermoregulation.

What are non-coding regulatory RNAs?

RNAs that regulate gene expression without encoding proteins; small RNAs can bind other RNAs through base pairing.

What two examples of non-coding regulatory RNA are given in the notes?

Small RNAs (sRNAs) and antisense RNAs (asRNAs).

What are riboswitches?

Structured regions within the 5′ untranslated region (5′ UTR) that bind small-molecule ligands and regulate gene expression.

What are the two parts of a riboswitch, and what does each do?

The aptamer domain binds the ligand; the expression platform undergoes a conformational change.

How can riboswitches regulate gene expression?

They can cause transcription termination through a terminator loop or inhibit translation by blocking the ribosome-binding site (RBS).

What ligand does the TPP riboswitch bind?

Thiamine pyrophosphate.

(Multiple choice) Which riboswitch domain binds the ligand? A. Expression platform B. Aptamer domain C. Ribosome-binding site D. Terminator poly-U sequence

B. Aptamer domain.

What is thermoregulation in gene expression, and what example is given?

Gene expression regulation in response to temperature changes; σ32 (rpoH), involved in the heat-shock response, is an example.

How can temperature regulate ribosome binding according to the notes?

Temperature changes control ribosome binding.

What are small regulatory RNAs (sRNAs)?

Non-coding RNAs that regulate gene expression through base pairing; they are approximately 50–200 nucleotides long.

What characterises trans-encoded sRNAs?

They are located elsewhere from their target, are less complementary to the target, and usually require chaperones to assist base pairing.

What characterises cis-encoded sRNAs?

They are located at the same location as their target, are antisense RNAs transcribed from the complementary strand, and exhibit extensive base pairing.

Compare trans-encoded and cis-encoded regulatory RNAs.

Trans-encoded RNAs are located elsewhere, have less complementarity, and usually need chaperones; cis-encoded antisense RNAs are transcribed from the complementary strand and have extensive base pairing.

What are the possible outcomes of sRNA regulation?

Inhibition or activation of transcription or translation, and promotion of mRNA stability or degradation.

What is MicF, and under what conditions is it induced?

A small regulatory RNA induced under stress conditions.

How does MicF regulate its target?

It binds and blocks the ribosome-binding site (RBS).

Which target does MicF control, and what is the outcome?

It controls OmpF, an outer membrane porin, reducing cell membrane permeability.