Regulation of Gene Expression & Molecular Genetics – Practice Flashcards

A comprehensive set of question-and-answer flashcards covering key concepts from bacterial and eukaryotic gene regulation, operon mechanics, molecular cloning, biotechnology tools, and genetic research strategies.

What two processes make up gene expression in the Central Dogma?

Transcription and translation.

What is constitutive transcription in bacteria?

Continuous gene expression with no regulatory control.

When does regulated transcription occur in bacteria?

Only under certain environmental conditions, such as nutrient availability.

In negative control of transcription, which protein binds DNA to block transcription?

A repressor protein.

In positive control of transcription, what must bind DNA to initiate transcription?

An activator protein.

What molecule removes a repressor from DNA in an inducible system?

An inducer.

What name is given to a small molecule that activates a repressor in a repressible system?

A corepressor.

What bacterial operon provides the classic example of both negative and positive control?

The lac operon of E. coli.

Which sugar is E. coli’s preferred energy source?

Glucose.

What three structural genes are found in the lac operon mRNA?

lacZ, lacY, and lacA.

What enzyme does lacZ encode and what is its role?

β-galactosidase; it cleaves lactose into glucose and galactose and converts lactose to allolactose.

What is the function of lacY?

It encodes lactose permease, which transports lactose into the cell.

What molecule serves as the effector (inducer) that inactivates the Lac repressor?

Allolactose.

Which protein complex provides positive control of the lac operon?

The CAP-cAMP complex.

How does low glucose affect cAMP levels and lac transcription?

Low glucose → high cAMP → CAP-cAMP forms → transcription up-regulated.

What condition produces the highest lac operon transcription?

Lactose present and glucose absent (inducer present, CAP-cAMP bound).

What effect does a loss-of-function mutation in lacI (lacI–) have on lac operon expression?

Constitutive expression (operon always ON).

What is an operator-constitutive (OC) mutation?

A mutation in lacO that prevents repressor binding, causing constitutive expression.

What is the phenotype of a super-repressor (lacIS) mutation?

Repressor cannot bind inducer; lac operon remains OFF even with lactose present.

If E. coli cannot grow on lactose-only plates, which lac mutation is most likely?

lacIS (super-repressor).

What regulatory gene lies adjacent to but outside the lac operon?

lacI.

Why do cells always have a tiny amount of β-galactosidase even without lactose?

Basal transcription of the lac operon.

What is the purpose of using IPTG in cloning vectors with lac operators?

IPTG mimics allolactose to induce expression of an inserted gene without being hydrolyzed.

Name the five structural genes in the trp operon.

trpE, trpD, trpC, trpB, and trpA.

What molecule acts as corepressor of the trp operon?

Tryptophan.

Is the trp operon mainly inducible or repressible?

Repressible (turned off when tryptophan is abundant).

What secondary regulatory mechanism fine-tunes trp operon transcription?

Attenuation via alternative mRNA hairpins in the leader (trpL) region.

Which hairpin terminates trp transcription early?

The 3–4 hairpin (terminator) when tryptophan is plentiful.

Which hairpin allows full trp operon transcription?

The 2–3 hairpin (antiterminator) when tryptophan is scarce.

What sigma factor drives heat-shock gene expression in E. coli?

Sigma 32 (σ32).

Which sigma factor is used for normal E. coli transcription at 37 °C?

Sigma 70 (σ70).

Give two key differences between prokaryotic and eukaryotic gene regulation.

Eukaryotes separate transcription & translation and package DNA into chromatin; prokaryotes usually regulate at transcription and lack a nucleus.

What are enhancers and silencers?

Distal regulatory DNA sequences that respectively increase or decrease transcription by interacting with promoter-bound proteins.

What is an insulator sequence?

A DNA element that blocks an enhancer from activating an unintended promoter.

Where is the limb-specific enhancer for the human Sonic Hedgehog (SHH) gene located?

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

Which yeast protein activates GAL gene transcription by binding UASG elements?

GAL4.

How is GAL4 kept inactive when galactose is absent?

GAL80 binds GAL4’s activation domain.

What proteins relieve GAL80 inhibition when galactose is present?

Galactose and GAL3, which bind GAL80 and release GAL4.

Why is the GAL4-UAS system useful in Drosophila genetics?

Flies lack endogenous GAL4 and UAS sites, allowing controlled ON/OFF gene expression with specific drivers and responders.

What enzyme processes double-stranded RNA into 21-24 bp fragments in RNAi?

Dicer.

What complex uses guide RNA to silence genes post-transcriptionally?

RISC (RNA-induced silencing complex).

Differentiate miRNA and siRNA.

miRNA originates from endogenous transcripts; siRNA comes from exogenous dsRNA such as viruses.

What are DNase I hypersensitive sites?

Regions of euchromatin that are easily digested, indicating active transcription.

What does chromatin immunoprecipitation (ChIP) detect?

DNA regions bound by a specific regulatory protein in vivo.

What is a restriction enzyme?

A nuclease that recognizes a specific DNA sequence and cuts both strands.

Why are EcoRI cuts said to produce sticky ends?

They leave single-stranded overhangs (5′-AATT).

How often is a 6-bp site like EcoRI expected to occur by chance?

Approximately once every 4^6 = 4096 bp.

How many fragments result when a circular plasmid is cut by an enzyme with three recognition sites?

Four fragments.

What enzyme forms phosphodiester bonds in recombinant DNA ligations?

DNA ligase.

What gene provides antibiotic selection in many cloning plasmids?

A β-lactamase gene conferring ampicillin resistance.

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

White (lacZ disrupted).

What are the three main steps of molecular cloning?

(1) Ligate DNA fragment into vector, (2) transform host cells, (3) replicate and select clones.

Why were methionine codons added to insulin fusion constructs in E. coli expression?

To allow cleavage with cyanogen bromide to separate insulin chains from β-galactosidase.

What is CRISPR-Cas9 primarily used for in genetics?

Genome editing via targeted double-strand breaks directed by guide RNA.

Define gene knockout.

Deletion or disruption of a gene’s coding region to abolish its function.

How does gene knockdown differ from knockout?

Knockdown reduces expression (e.g., via RNAi) without deleting the gene.

What is a reporter gene?

A detectable gene (e.g., GFP, lacZ) fused to regulatory sequences to monitor gene expression.

What is enhancer trapping?

Random insertion of a reporter with weak promoter; expression occurs only if near an endogenous enhancer.

How can GAL80 be used to refine GAL4 expression patterns?

GAL80 expression in specific tissues blocks GAL4 activity there, preventing UAS-gene expression locally.

What is GAL80^TS and why is it useful?

A temperature-sensitive GAL80 allowing temporal control of GAL4 activity by shifting temperature.

What is optogenetics?

Controlling neuronal activity by expressing light-sensitive ion channels (e.g., ChR2) via GAL4-UAS and stimulating with light.

Define forward genetics.

Identifying genes responsible for phenotypes by screening mutants produced with mutagens.

What is the purpose of a complementation test in genetics?

To determine whether two recessive mutations affect the same gene or different genes.

List three features that make E. coli a model organism.

Short 20-min generation time, single small chromosome, easy lab cultivation.

Which model organism was first to have its complete connectome mapped?

Caenorhabditis elegans.

Name two core circadian clock genes in Drosophila.

period (per) and timeless (tim).

What transcription factors activate per and tim expression?

CLK (Clock) and CYC (Cycle).

Which blue-light receptor resets the Drosophila clock by degrading TIM?

Cryptochrome (CRY).

How does royal jelly influence honey-bee caste development?

By reducing DNA methylation, activating queen-specific gene expression.

What class of proteins assists in folding or degrading heat-damaged proteins?

Heat shock proteins (Hsps).

Which transcription factor regulates Hsp expression?

Heat shock factor 1 (HSF1).