Topic 6: DNA Replication, Gene Control, and DNA Mutation

Comprehensive practice flashcards for BIO152 Cell Biology (Topic 6), covering DNA replication mechanisms and enzymes, prokaryotic and eukaryotic gene control (including the lac operon), and DNA and chromosomal mutations.

What is the central dogma of molecular biology?

The directional flow of genetic information from $\text{DNA} \rightarrow \text{RNA} \rightarrow \text{Protein}$, mediated by transcription and translation.

During which phase of the eukaryotic cell cycle does DNA replication take place, and why?

It occurs during the $\text{S}$ phase (synthesis phase) of interphase to duplicate genetic material before the cell enters the $\text{M}$ (mitotic) phase, ensuring each daughter cell receives an identical set of DNA.

Where does DNA replication occur within eukaryotic cells versus prokaryotic cells?

In eukaryotes, DNA replication takes place in the nucleus; in prokaryotes, it takes place in the nucleoid region of the cytoplasm.

Which types of chemical bonds link individual nucleotides along a single strand, and which hold complementary base pairs together in the double helix?

Phosphodiester bonds link adjacent sugars and phosphate groups along the backbone, whereas hydrogen bonds hold complementary base pairs ($\text{A}$ with $\text{T}$, and $\text{C}$ with $\text{G}$) together between the two antiparallel strands.

Which of the three proposed models for DNA replication shown in the diagram was proven to be correct by Meselson and Stahl (1958)?

Semiconservative replication, in which each replicated duplex consists of one intact parental strand and one newly synthesized strand.

What are the four main stages of DNA replication in prokaryotes?

1. Initiation\n2. Primer binding\n3. Elongation\n4. Termination

What is the function of DNA helicase during the initiation of replication, and how fast does it operate?

DNA helicase unwinds the double helix at the origin of replication by breaking hydrogen bonds between complementary base pairs, spinning at approximately $10\,000\,\text{rpm}$.

What role do single-stranded DNA-binding proteins (SSBs) play at the replication fork?

SSBs bind to and stabilize the exposed single-stranded template strands to prevent them from re-annealing into a double helix.

How does topoisomerase prevent DNA breakage ahead of the replication fork?

It acts as a swivel to relieve supercoiling tension generated by helicase unwinding by cutting, uncoiling, and rejoining the DNA backbone.

Why is the enzyme primase necessary to initiate DNA synthesis?

DNA polymerases can only add nucleotides to an existing $3'\text{-OH}$ group; primase synthesizes a short complementary RNA primer (approximately $10\,\text{bases}$ long) to provide this free $3'\text{-OH}$ group.

In which direction does DNA polymerase III synthesize DNA, and what building blocks does it incorporate?

It synthesizes strictly in the $5' \rightarrow 3'$ direction by adding deoxyribonucleoside triphosphates ($\text{dNTPs}$: $\text{dATP}$, $\text{dGTP}$, $\text{dCTP}$, $\text{dTTP}$) to the free $3'\text{-OH}$ end via phosphodiester bonds.

How does synthesis of the leading strand differ from synthesis of the lagging strand at a replication fork?

The leading strand is synthesized continuously in the $5' \rightarrow 3'$ direction toward the unwinding replication fork, while the lagging strand is synthesized discontinuously away from the fork in short fragments called Okazaki fragments.

What enzymatic property allows DNA polymerase to maintain high fidelity and proofread newly added nucleotides?

Its $3' \rightarrow 5'$ exonuclease activity, which detects mismatched base pairings, removes the incorrect nucleotide from the $3'$ end, and replaces it with the correct complementary nucleotide.

What are the specific functions of DNA polymerase I and DNA ligase during the termination of prokaryotic DNA replication?

DNA polymerase I removes the RNA primers and replaces them with deoxyribonucleotides, and DNA ligase seals the remaining nicks by synthesizing phosphodiester bonds to join adjacent fragments into a continuous strand.

How does Polymerase Chain Reaction (PCR) amplify target DNA sequences across cycles?

PCR amplifies target sequences exponentially in vitro by repeatedly cycling through replication steps, yielding product molecules equal to $2^n$, where $n$ is the number of cycles completed.

What is the operational distinction between constitutive genes and inducible genes?

Constitutive genes (such as housekeeping genes encoding ribosomal proteins) are continuously transcribed under all conditions, whereas inducible genes are transcribed only when specific environmental signals or substrates are present.

What is an operon, and does the bacterial lac operon produce monocistronic or polycistronic mRNA?

An operon is a cluster of genes regulated under a single shared promoter; the lac operon produces polycistronic (multigenic) mRNA containing the coding sequences for multiple structural proteins.

What are the three structural genes of the lac operon and the specific proteins they encode?

1. lacZ encodes $\beta$-galactosidase\n2. lacY encodes galactoside permease\n3. lacA encodes transacetylase

What are the physiological functions of galactoside permease and $\beta$-galactosidase in lactose metabolism?

Galactoside permease transports lactose across the bacterial plasma membrane into the cell, and $\beta$-galactosidase hydrolyzes the disaccharide lactose into the monosaccharides galactose and glucose.

What formatting convention distinguishes bacterial genes from their protein products?

Gene symbols are italicized, lowercase, and end in a capital letter (e.g., lacY), whereas protein names are non-italicized with capital first and last letters (e.g., LacY).

What is the structural and functional distinction between a promoter and an operator?

A promoter is the DNA sequence where RNA polymerase binds to initiate transcription, whereas an operator is the regulatory DNA sequence where regulatory proteins (such as the LacI repressor) bind to control access to the promoter.

How does negative regulation shut down transcription of the lac operon in the absence of lactose?

The regulatory gene lacI produces the active LacI repressor protein, which binds directly to the operator, creating a physical roadblock that prevents RNA polymerase from moving downstream and transcribing the operon.

How is negative regulation of the lac operon relieved in the presence of lactose?

Allolactose (an isomer of lactose) binds to the LacI repressor protein, inducing an allosteric conformational change that inactivates the repressor so it can no longer bind to the operator, allowing RNA polymerase to proceed.

What molecular mechanism mediates positive regulation of the lac operon under low or absent glucose conditions?

Adenylyl cyclase remains active and converts $\text{ATP}$ to $\text{cAMP}$. $\text{cAMP}$ binds to the catabolite activator protein ($\text{CAP}$, or $\text{CRP}$) to form the $\text{CAP-cAMP}$ complex, which binds to the $\text{CAP}$ site near the promoter and recruits RNA polymerase to bind efficiently.

What is catabolite repression, and how does glucose exert this effect on the lac operon?

Catabolite repression is the repression of operons for alternative sugars when a preferred catabolite (glucose) is available; glucose inhibits adenylyl cyclase, lowering $\text{cAMP}$ levels, preventing $\text{CAP-cAMP}$ formation, and leaving RNA polymerase unable to efficiently bind the promoter.

Why is catabolite repression biologically advantageous for Escherichia coli?

It prevents the unnecessary expenditure of cellular energy on transcribing and translating enzymes to break down secondary sugars when glucose is already present to fuel metabolism.

Why is the induction of the lac operon described as not being an 'all-or-nothing' response?

Even in the absence of an inducer, basal 'leaky' transcription produces a minimal level of permease and $\beta$-galactosidase ($< 10\,\text{copies}$ per cell), ensuring initial lactose molecules can enter the cell to trigger full induction (up to $1000\times$).

What are the five distinct levels of eukaryotic gene regulation?

1. Genomic level\n2. Transcriptional level\n3. Post-transcriptional level\n4. Translational level\n5. Post-translational level

In the shown transmission electron micrograph (TEM) of a eukaryotic nucleus, what structural and functional difference exists between euchromatin and heterochromatin?

Euchromatin (the lighter regions) is loosely packed, decondensed chromatin actively undergoing transcription, whereas heterochromatin (the dark, dense regions) is tightly condensed chromatin that is transcriptionally inactive.

How do enhancers and silencers control gene expression at the transcriptional level in eukaryotes?

Enhancers bind activator proteins to stimulate and increase transcription at the promoter, while silencers bind repressor proteins to inhibit and decrease transcription.

What modifications occur during the post-transcriptional processing of a eukaryotic primary transcript (pre-mRNA)?

Addition of a $5'$ cap, addition of a $3'$ poly(A) tail, and RNA splicing where non-coding intervening sequences (introns) are excised and coding sequences (exons) are spliced together to form mature mRNA.

Based on the illustrated mechanism, how does iron concentration regulate ferritin synthesis at the translational level?

At low iron concentration, the active IRE-binding protein binds the iron-response element (IRE) in the $5'$ untranslated region of ferritin mRNA, physically blocking ribosome progression; at high iron concentration, iron binds the IRE-binding protein, causing it to dissociate and allowing translation of ferritin.

According to the illustrated pathway, how is glycogen phosphorylase regulated at the post-translational level?

Phosphorylase kinase uses $2\,\text{ATP}$ to phosphorylate inactive glycogen phosphorylase b, converting it into active glycogen phosphorylase a; phosphorylase phosphatase removes the phosphate groups by dephosphorylation, returning it to the inactive b form.

What is a DNA mutation, and why are mutation events relatively rare in cells?

A DNA mutation is an alteration in the nucleotide sequence. Mutations are rare because DNA forms a geometrically tight double helix where bases are sheltered inside the sugar-phosphate backbone, and DNA polymerase proofreading detects and repairs errors.

If the mutation rate in a bacterial culture is $1\,\text{in}\,10^8\,\text{cells/gene}$, how many mutants per gene are expected in a colony containing $10^9\,\text{cells}$?

$\frac{10^9\,\text{cells}}{10^8\,\text{cells/gene}} = 10\,\text{mutants per gene}$.

What distinguishes spontaneous (non-induced) mutations from induced mutations?

Spontaneous mutations occur naturally and randomly without external influence (typically due to uncorrected replication errors), whereas induced mutations result from exposure to external physical, chemical, or biological mutagens.

What are representative examples of chemical, physical, and biological mutagens cited in the lectures?

• Chemical: Nitrosoguanidine (NSG) and colchicine\n- Physical: Ultraviolet (UV) light, X-rays, and gamma rays\n- Biological: Viruses

What is the biological difference between a somatic mutation and a germinal mutation?

Somatic mutations occur in non-gametic body cells, affect only the individual (e.g., skin cancer), and cannot be passed to offspring; germinal mutations occur in reproductive cells (egg or sperm), are inherited by offspring, and affect every cell in the resulting organism (e.g., sickle-cell disease, cystic fibrosis).

At the DNA level, what is the defining structural difference between a transition mutation and a transversion mutation?

A transition is a substitution between bases of the same chemical class (purine $\leftrightarrow$ purine: $\text{A} \leftrightarrow \text{G}$, or pyrimidine $\leftrightarrow$ pyrimidine: $\text{C} \leftrightarrow \text{T}$); a transversion is a substitution between bases of different classes (purine $\leftrightarrow$ pyrimidine: $\text{A}/\text{G} \leftrightarrow \text{C}/\text{T}$).

What are the four primary phenotypic classifications of point mutations at the protein level?

1. Silent mutation (codon changes but codes for the same amino acid)\n2. Missense mutation (codon changes to specify a different amino acid)\n3. Nonsense mutation (codon changes to a premature stop codon, truncating the protein)\n4. Frameshift mutation (insertion or deletion shifts the reading frame, altering all downstream amino acids)

In the comparison between normal and sickle-cell haemoglobin shown, what exact mutation occurs at the DNA level and at the protein level for codon 6 ($\text{GAG} \rightarrow \text{GTG}$)?

At the DNA level, it is a transversion mutation ($\text{A} \rightarrow \text{T}$, swapping a purine for a pyrimidine); at the protein level, it is a missense mutation (replacing glutamic acid with valine, $\text{Glu} \rightarrow \text{Val}$).

What is the distinction between aneuploidy and polyploidy in chromosomal mutations?

Aneuploidy involves an abnormal number of individual chromosomes within a set (e.g., trisomy 21 in Down syndrome or monosomy X in Turner syndrome); polyploidy involves the presence of one or more entire extra chromosome sets (e.g., triploidy [$3n$], which in humans typically results in miscarriage).

What specific type of structural chromosome mutation is represented when segment order A-B-C-D-E-F is rearranged to A-E-D-C-B-F?

A chromosomal inversion, in which an internal chromosome segment (B-C-D-E) is cleaved, rotated $180^\circ$, and reinserted into the chromosome.

Which specific chromosomal structural mutations cause Cri du chat syndrome and Burkitt's lymphoma?

Cri du chat syndrome is caused by a chromosomal deletion (loss of part of chromosome 5); Burkitt's lymphoma is caused by a reciprocal chromosomal translocation (e.g., exchange of segments between chromosomes 8 and 14).

When transcribing a gene, how does RNA polymerase interpret template versus coding strands?

RNA polymerase reads the template strand in the $3' \rightarrow 5'$ direction to synthesize mRNA in the $5' \rightarrow 3'$ direction, incorporating uracil ($\text{U}$) instead of thymine ($\text{T}$); the resulting mRNA sequence matches the non-template (coding) strand, except that $\text{T}$ is replaced by $\text{U}$.