Regulation of Gene Expression and Recombinant DNA – Comprehensive Review

A set of 200 question-and-answer flashcards covering bacterial and eukaryotic gene regulation, operon mechanisms, recombinant DNA technology, genetic engineering tools, and model organism strategies.

What is the Central Dogma of molecular biology?

Information flows from DNA to RNA to protein through transcription and translation.

Which two cellular processes together constitute gene expression?

Transcription and translation.

What is constitutive transcription in bacteria?

Continuous, unregulated expression of a gene regardless of environmental conditions.

What characterizes regulated transcription in bacteria?

Gene expression that occurs only under specific environmental conditions such as nutrient availability.

In negative control of transcription, which molecule binds DNA to prevent transcription?

A repressor protein.

In positive control of transcription, which molecule binds DNA to initiate transcription?

An activator protein.

What is an allosteric change?

A conformational change in a protein caused by binding of a molecule at a site other than the active site, altering protein function.

How does an inducer affect a repressor in negative control systems?

It binds the repressor, causes an allosteric change, and releases the repressor from DNA to allow transcription.

What is a corepressor?

A small molecule that binds a repressor protein, enabling it to attach to DNA and inhibit transcription.

What happens when a corepressor binds its repressor?

The corepressor-repressor complex binds the operator and blocks transcription.

In positive control, what is an effector molecule?

A small molecule that binds an activator protein, enabling it to bind DNA and stimulate transcription.

What is the role of an allosteric inhibitor in positive control?

It binds an activator protein, causing the activator to detach from DNA and shut off transcription.

In which organism was the lac operon discovered?

Escherichia coli (E. coli).

Under what environmental conditions is the lac operon maximally expressed?

When lactose is present and glucose is absent.

What sugar is the preferred energy source for E. coli?

Glucose.

Which lac operon enzyme transports lactose into the cell?

Lac permease (product of lacY).

Which lac operon enzyme cleaves lactose into glucose and galactose?

β-galactosidase (product of lacZ).

Which molecule acts as the natural inducer of the lac operon?

Allolactose.

Is the lac operon transcript monocistronic or polycistronic?

Polycistronic; one mRNA encodes multiple proteins.

Name the three structural genes of the lac operon.

lacZ, lacY, and lacA.

Which gene encodes the lac repressor protein?

lacI.

Where does the lac repressor bind to block transcription?

The operator sequence (lacO).

What does CAP stand for in the lac system?

Catabolite activator protein.

Which second messenger molecule binds CAP?

Cyclic adenosine monophosphate (cAMP).

How does glucose concentration affect cAMP levels?

Low glucose increases cAMP; high glucose lowers cAMP.

What is the lac operon transcription level when glucose is low and lactose is present?

High (fully induced).

What happens to lac operon transcription when glucose is high and lactose is absent?

No transcription; the operon is repressed.

What term describes the small amount of lac expression even under repression?

Basal transcription.

What effect does a loss-of-function mutation in lacI (lacI⁻) have?

Constitutive expression of the lac operon.

What is an operator-constitutive (OC) mutation?

A mutation in the operator that prevents repressor binding, leading to constitutive operon expression.

What is a lacIS (super-repressor) mutation?

A repressor mutation that cannot bind inducer, causing permanent repression of the operon.

Which mutation most likely prevents growth on lactose-only media despite functional β-galactosidase gene copies?

A super-repressor mutation (lacIS).

What DNA configuration can form when the lac repressor binds two operator sites?

A DNA loop that enhances repression.

Why is the lac operon useful in plasmid vectors?

It provides an inducible ON/OFF switch for transgene expression via IPTG or allolactose analogs.

What chemical analog of allolactose is frequently used to induce lac-controlled genes in the lab?

IPTG (isopropyl β-D-1-thiogalactopyranoside).

Which amino acid is synthesized by the trp operon?

Tryptophan.

How many structural genes compose the trp operon?

Five (trpE, trpD, trpC, trpB, trpA).

What is the promoter of the trp operon called?

trpP.

What is the operator of the trp operon called?

trpO.

What is trpL in the trp operon?

A leader sequence (attenuator) preceding the structural genes that controls attenuation.

Which gene encodes the trp repressor protein?

trpR.

When tryptophan is abundant, what complex forms to repress the trp operon?

Tryptophan acts as a corepressor by binding the trp repressor, allowing it to bind the operator.

Is the trp operon controlled by negative or positive regulation?

Negative control via a corepressor-activated repressor.

What is attenuation in the trp operon?

A second regulatory mechanism using leader-region RNA hairpins to terminate or continue transcription based on tryptophan levels.

Which RNA secondary structure is central to attenuation?

Hairpin (stem-loop) structures in the leader mRNA.

How many consecutive tryptophan codons are in the trpL peptide?

Two.

Which hairpin (3-4 or 2-3) causes premature termination of trp transcription?

The 3-4 hairpin.

Under low tryptophan, which hairpin forms to allow full transcription?

The 2-3 antiterminator hairpin.

What is the heat-shock response in bacteria?

Transcriptional activation of chaperone and protease genes when temperature exceeds 37 °C.

Which sigma factor mediates E. coli heat-shock gene transcription?

Sigma 32 (σ32).

Where does transcription occur in eukaryotic cells?

Inside the nucleus.

Name two distal DNA elements that regulate eukaryotic transcription.

Enhancers and silencers.

What is chromatin remodeling?

Enzymatic alteration of chromatin structure to regulate accessibility of DNA to transcription machinery.

DNA methylation is generally associated with what effect on gene expression?

Repression or silencing.

Define an enhancer sequence.

A regulatory DNA element that increases transcription of a gene from a distance when bound by activators.

Which gene exemplifies tissue-specific enhancer action in mammals?

Sonic hedgehog (SHH).

Approximately how far upstream is the SHH limb enhancer located?

About 1 million base pairs upstream in an intron of another gene.

Which protein binds the UAS elements in yeast to activate galactose genes?

GAL4 protein.

Which protein inhibits GAL4 activity in the absence of galactose?

GAL80.

What is the state of GAL4 when galactose is absent?

It is bound by GAL80 and inactive.

Which protein helps release GAL4 upon galactose binding?

GAL3, together with galactose, binds GAL80 to free GAL4.

Which animal model commonly uses the GAL4-UAS system for gene control?

Drosophila melanogaster (fruit fly).

What is the primary function of insulator sequences?

To block or redirect enhancer activity, often by forming chromatin loops.

DNase I hypersensitivity typically indicates what chromatin state?

Euchromatin that is transcriptionally active.

Which enzyme processes double-stranded RNA into 21–24 bp fragments?

Dicer.

What does RISC stand for?

RNA-induced silencing complex.

In RISC, what is the guide strand?

The remaining RNA strand that base-pairs with complementary mRNA to mediate silencing.

List three possible outcomes after RISC binds its target.

mRNA degradation, translational inhibition, or chromatin modification to silence transcription.

What is a restriction enzyme?

A nuclease that recognizes a specific DNA sequence and cuts both strands at or near that site.

What are sticky ends?

Single-stranded overhangs left after restriction enzyme cutting, facilitating ligation with complementary ends.

A 6-bp recognition site is expected to occur once every how many bases?

4^6 = 4096 base pairs.

How many DNA fragments result when a circular plasmid is cut at three sites by one enzyme?

Four fragments.

Which technique separates DNA fragments by size?

Gel electrophoresis.

Define molecular cloning.

Inserting a DNA fragment into a vector and propagating it in a biological host to create identical copies.

Which enzyme covalently joins DNA fragments during cloning?

DNA ligase.

What is bacterial transformation?

Introduction of foreign DNA (usually a plasmid) into bacterial cells.

Which gene in plasmids commonly confers antibiotic resistance for selection?

β-lactamase (ampicillin resistance).

In blue-white screening, what color are colonies with functional lacZ?

Blue.

In blue-white screening, what colony color indicates successful insert integration?

White.

Why might E. coli fail to express certain eukaryotic proteins correctly?

Because it lacks eukaryotic post-translational modification machinery.

Which human hormone was first mass-produced using recombinant E. coli?

Insulin.

What is a fusion protein?

A single polypeptide encoded by two or more gene segments joined in frame.

Which chemical cleaves proteins at methionine residues during insulin production?

Cyanogen bromide.

What is a transgenic animal?

An organism whose genome has been altered to carry foreign DNA sequences.

What was the first cloned mammal called?

Dolly the sheep.

Does Dolly contain mitochondrial DNA from the nucleus donor?

No; mitochondria come from the egg donor.

Define forward genetics.

Identifying genes responsible for a phenotype by first finding mutants with that phenotype.

Define reverse genetics.

Starting with a known gene, altering it, and examining the resulting phenotype.

Name two reverse-genetic mutagenesis tools.

CRISPR-Cas9 gene editing and RNA interference (RNAi).

What is CRISPR-Cas9?

A genome-editing technology using Cas9 nuclease guided by RNA to make targeted DNA double-strand breaks.

What is a guide RNA in CRISPR?

An engineered RNA molecule that directs Cas9 to a specific genomic sequence by base pairing.

Which repair pathway often introduces indels after CRISPR cleavage?

Non-homologous end joining (NHEJ).

What is a gene knockout?

Complete inactivation or deletion of a gene’s coding sequence.

What is gene knockdown?

Partial reduction of gene expression, often via RNAi.

What is a reporter gene?

A gene whose product is easily detected and used to monitor gene expression or protein localization.

Give a common fluorescent reporter gene example.

Green fluorescent protein (GFP).

What is enhancer trapping?

Random insertion of a transgene with a weak promoter that becomes activated when near an endogenous enhancer.

In the GAL4-UAS system, what is the “driver” line?

A line expressing GAL4 under control of a chosen promoter.

In the GAL4-UAS system, what is the “responder” line?

A line with a gene of interest downstream of UAS sequences.

Why is UAS inactive in flies lacking the GAL4 protein?

Endogenous Drosophila transcription factors do not recognize UAS, so it requires GAL4 binding to activate transcription.