Molecular Biology Lecture I-V – Comprehensive Review

Question-and-Answer flashcards covering key concepts from Molecular Biology lectures: macromolecules, protein and nucleic-acid structure, gene expression, enzymology, replication, transcription, translation, biotechnology techniques, and advanced methods.

What does molecular biology study at its core?

Biological phenomena at the molecular level, especially nucleic acids, the information they contain, and the molecular basis of gene expression and regulation.

What common construction principle do proteins, nucleic acids and some polysaccharides share?

They are polymers assembled from repeating small units (monomers) that possess a common backbone and a variable side‐chain that gives specificity.

Which functional groups define an α-amino acid?

An amino group (-NH₂) and a carboxyl group (-COOH) both attached to the same alpha-carbon.

Why are almost all natural amino acids L-isomers?

Protein synthesis machinery recognizes only L-amino acids; the stereochemistry influences overall protein structure and function.

What reaction links amino acids in proteins?

A condensation reaction between the carboxyl of one amino acid and the amino of the next, forming a rigid, planar peptide (amide) bond and releasing water.

Which bond in a polypeptide confers rotational freedom and thus flexibility?

The bond at the α-carbon (Cα) adjacent to the rigid peptide bond.

Define the four levels of protein structure.

Primary: amino-acid sequence; Secondary: local regular folding (α-helix, β-sheet); Tertiary: overall 3-D folding of one polypeptide; Quaternary: association of multiple polypeptide chains.

What stabilises α-helices and β-sheets?

Hydrogen bonds between backbone carbonyl oxygens and amide hydrogens.

What are intrinsically disordered proteins?

Proteins or protein segments lacking a stable 3-D fold until they bind partners; ~1/3 of natural proteins contain such regions.

What experimental proof first indicated DNA carries genetic information in bacteria?

Griffith's transformation experiment with smooth/rough Streptococcus and mice (1928).

State Chargaff’s rule.

In DNA the amount of purines equals the amount of pyrimidines: [A]=[T] and [G]=[C].

Which two key pieces of evidence led Watson & Crick to propose the double helix?

Chargaff’s base ratios and Rosalind Franklin’s X-ray diffraction pattern showing a helical repeat (3.4 Å per base, 34 Å per turn).

Why does DNA have major and minor grooves?

The glycosidic bond geometry creates asymmetric backbones, leaving two unequal surface grooves along the helix.

How many hydrogen bonds link A–T and G–C base pairs respectively?

A–T have two H-bonds; G–C have three H-bonds (hence stronger).

What is DNA melting (denaturation)?

Separation of the double strands when hydrogen bonds are disrupted by heat or extreme pH, monitored by increased absorbance at 260 nm.

Define a palindromic DNA sequence and its structural consequence when single-stranded.

A sequence that reads the same 5'→3' on both strands; when single-stranded it can fold into a hairpin via internal complementarity.

List three major structural differences between DNA and RNA.

RNA has ribose (2'-OH), uses uracil instead of thymine, and is usually single-stranded and structurally versatile.

Name two common modified bases found in tRNA.

Inosine and pseudouridine (among many others).

What is negative DNA supercoiling and why is it useful?

Under-winding of the double helix; it facilitates local strand separation required for replication and transcription.

Differentiate topoisomerase I and II.

Topo I cuts one strand to relax supercoils (ΔLk = ±1); Topo II cuts both strands, can introduce or remove supercoils and decatenate DNA (ΔLk = ±2).

Give an example of an antibiotic targeting bacterial topoisomerases.

Fluoroquinolones (e.g., ciprofloxacin) poison DNA gyrase/topo IV.

What proteins package eukaryotic DNA into ‘beads-on-a-string’?

Histones (octamer of H2A, H2B, H3, H4) forming nucleosomes; H1 associates with linker DNA.

How can histone tail acetylation influence chromatin?

Neutralises Lys positive charges, weakens DNA-histone interaction, leading to a more open, transcriptionally active chromatin.

What experiment proved semiconservative DNA replication?

Meselson–Stahl density-gradient experiment with ¹⁵N/¹⁴N E. coli DNA (1958).

Name the three proposed DNA replication models tested by Meselson & Stahl.

Conservative, dispersive, and semiconservative (the correct one).

Which enzyme synthesises RNA primers during replication?

Primase (an RNA polymerase).

Compare the roles of DNA polymerase III and DNA polymerase I in E. coli.

Pol III: main, highly processive replicative polymerase; Pol I: removes RNA primers via 5'→3' exonuclease and fills gaps (nick translation).

What are Okazaki fragments?

Short DNA fragments (~1000 nt in bacteria, ~100–200 nt in eukaryotes) synthesised discontinuously on the lagging strand.

What is the function of the sliding clamp (β-clamp in bacteria)?

Encircles DNA, tethering DNA polymerase to the template to ensure high processivity.

Which bond does DNA ligase create and what cofactor does it utilise?

A phosphodiester bond between adjacent 3’-OH and 5’-P; uses ATP (or NAD⁺ in some bacteria) via a ligase-AMP intermediate.

State two key properties of the genetic code revealed by Nirenberg & Khorana.

It is read in non-overlapping triplets and is degenerate (most amino acids encoded by multiple codons).

Which codon serves as both start and Met codon in standard code?

AUG (initiates translation with formyl-methionine in prokaryotes, Met in eukaryotes).

What enzyme charges tRNAs and how does it ensure accuracy?

Aminoacyl-tRNA synthetase; uses specific recognition of tRNA identity elements and proofreading (kinetic and chemical) to keep error rate ~10⁻⁴–10⁻⁵.

Explain wobble pairing and its molecular basis.

Flexibility at the tRNA anticodon 1st position (5’ end) allows non-Watson-Crick pairing—often via inosine—enabling one tRNA to read several synonymous codons.

What are the three tRNA binding sites on the ribosome?

A (aminoacyl), P (peptidyl), and E (exit) sites.

Which rRNA performs peptidyl-transferase activity?

23S rRNA (prokaryotes) / 28S rRNA (eukaryotes) acts as a ribozyme in the large subunit.

Name an antibiotic that blocks the ribosomal A-site during translation.

Tetracycline (binds 30S and prevents aminoacyl-tRNA entry).

What is the Shine–Dalgarno sequence?

A purine-rich ribosome binding site in prokaryotic mRNA that base-pairs with 16S rRNA to position the start codon in the P site.

Which factors mediate translation termination in bacteria?

Release factors RF1, RF2 recognise stop codons, RF3 promotes peptide release and ribosome recycling.

Give one important difference between prokaryotic and eukaryotic mRNA initiation signals.

Eukaryotes rely on 5’ cap recognition and scanning (Kozak sequence), whereas prokaryotes use Shine–Dalgarno alignment.

What is PCR and which enzyme made it practical?

Polymerase Chain Reaction: exponential in vitro amplification of DNA using primers and thermostable Taq DNA polymerase (from Thermus aquaticus).

Describe the three main temperature steps in a PCR cycle.

Denaturation (~95 °C), primer annealing (~50–65 °C), extension (~72 °C).

What modification turns PCR into RT-PCR?

Addition of reverse transcriptase to first convert RNA into complementary DNA (cDNA) before amplification.

How does qPCR allow quantification of starting template?

Uses fluorescence (e.g., SYBR Green or probe) monitored each cycle; the threshold cycle (Ct) inversely correlates with initial template amount.

Which two enzymes are essential for creating recombinant DNA in cloning?

Type II restriction endonucleases to cut DNA at specific sites and DNA ligase to join compatible ends.

What three core features must a plasmid cloning vector possess?

Origin of replication (Ori), selectable marker (e.g., antibiotic resistance), and multiple cloning site (MCS).

Why do blue/white colonies indicate successful insertion in pUC-type vectors?

Insertion disrupts the lacZ α-fragment; colonies with insert cannot cleave X-gal and remain white, whereas empty vectors produce blue colonies.

Which PCR method is used to introduce defined mutations into DNA?

Site-directed mutagenesis via primers containing the desired nucleotide change.

What property of DNA allows separation by agarose gel electrophoresis?

Uniform negative charge per unit length from phosphate backbone causes size-dependent migration towards the anode through the gel matrix.

How are DNA fragments visualised in gels?

Intercalating dyes (e.g., ethidium bromide, SYBR Safe) fluoresce under UV/blue light when bound to double-stranded DNA.

What is a Southern blot used for?

Detection of specific DNA fragments by transferring gel-separated DNA to a membrane and probing with a labelled complementary sequence.

Explain the principle of SDS-PAGE.

SDS denatures proteins and imparts uniform negative charge; polyacrylamide gel separates them by size under an electric field.

Which blotting technique detects specific proteins and what probes are used?

Western blot; uses primary antibodies against the protein and enzyme- or fluor-conjugated secondary antibodies for detection.

What does ChIP-seq allow researchers to map?

Genome-wide binding sites of a DNA-associated protein in living cells by chromatin immunoprecipitation followed by next-generation sequencing.

Define a transgenic organism.

An organism whose genome has been altered by insertion, deletion, or modification of genes via recombinant DNA technologies.

Differentiate knockout and knock-in mouse models.

Knockout: targeted deletion/inactivation of a gene; Knock-in: targeted insertion or replacement to add or modify a gene.

What is GFP and why is it valuable in molecular biology?

Green Fluorescent Protein from Aequorea victoria; its gene can be fused to proteins or expressed in organisms to visualise cells and gene expression in vivo.

Which mobile genetic elements move via an RNA intermediate?

Retrotransposons (class I transposons) transpose through an RNA copy and reverse transcription.

What is the function of DNA methylation at bacterial origins regarding replication frequency?

Hemimethylated origins after replication prevent immediate re-initiation; only fully methylated origins can fire again, ensuring once-per-cycle replication.

Name two widely used next-generation sequencing platforms.

Illumina (sequencing-by-synthesis with reversible terminators) and Oxford Nanopore (single-molecule pore current detection).

What is an isopycnic density-gradient centrifugation used for?

Separating molecules (e.g., DNA, organelles) solely on the basis of buoyant density until each reaches an equilibrium position in the gradient.

Why is HRP-conjugated secondary antibody common in ELISA and Western blot?

Horseradish peroxidase catalyses chemiluminescent or colorimetric reactions, producing a sensitive, easily detectable signal for bound antibodies.