Molecular Biology: DNA, RNA, and Central Dogma

• DNA is a stable, double-stranded helical molecule composed of deoxyribonucleotides, which include a phosphate group, deoxyribose sugar, and nitrogenous bases (adenine, guanine, cytosine, or thymine).
• RNA is a single-stranded molecule containing ribose sugar and uracil in place of thymine, serving in genetic information transfer (mRNA), protein synthesis (rRNA, tRNA), and gene regulation (miRNA).
• The central dogma outlines genetic information flow: DNA to RNA via transcription, then RNA to protein via translation.
• Transcription involves RNA polymerase creating a complementary RNA strand from a DNA template.
• Translation involves ribosomes assembling amino acids into a protein based on the RNA sequence, using tRNA.
• Reverse transcription, used by retroviruses, converts RNA back into DNA with reverse transcriptase for integration into the host genome.
• DNA replication starts at the origin of replication (oriC), where helicase unwinds the helix and primase creates RNA primers.
• DNA polymerase III adds nucleotides in the 5’ to 3’ direction during elongation.
• The leading strand is synthesized continuously, while the lagging strand is synthesized in Okazaki fragments.
• DNA polymerase III uses its 3’ to 5’ exonuclease activity for proofreading and error correction.
• Replication terminates at specific sequences, DNA strands separate, and DNA ligase joins Okazaki fragments.
• Topoisomerases relieve tension from DNA supercoiling, with DNA gyrase crucial in bacteria for preventing excessive coiling.
• Plasmids are extrachromosomal DNA carrying beneficial genes like antibiotic resistance.
• Pathogens exploit host cells, often using virulence factors from horizontally transferred genes.
• Photosynthesis and nitrogen fixation are genetically regulated in bacteria like cyanobacteria and nitrogen-fixing bacteria, respectively.
• Nitrogen-fixing bacteria convert atmospheric nitrogen (N₂) into ammonia (NH₃), which is essential for ecosystem nutrient cycles.
• Transcription synthesizes RNA from a DNA template via RNA polymerase during initiation, elongation, and termination with RNA synthesized 5’ to 3’.
• Initiation involves RNA polymerase binding to the promoter sequence guided by a sigma factor.
• Sigma factors direct RNA polymerase to specific promoter regions to initiate transcription, with different sigma factors responding to environmental changes.
• Sigma factor 70 (σ⁷⁰) regulates housekeeping genes for essential cell functions, controlling gene expression by promoter accessibility.
• RNA synthesis occurs in the 5’ to 3’ direction, reading the DNA template 3’ to 5’.
• Rho-dependent termination involves the Rho protein disrupting transcription, while Rho-independent termination involves a GC-rich hairpin loop followed by a poly-U sequence.
• mRNA encodes proteins with a short half-life (3–5 minutes) and contains codons for amino acid sequences.
• rRNA forms the structural and functional components of ribosomes, with three types (16S, 23S, and 5S rRNA), that have long half-lives.
• tRNA carries amino acids to ribosomes, featuring an anticodon end for mRNA pairing and an acceptor end for amino acid binding, and has a long half-life.
• sRNA regulates transcription, translation, and RNA stability, aiding bacterial response to stress and metabolic changes.
• tmRNA rescues ribosomes stalled on damaged mRNA; it has properties of both tRNA and mRNA. Ribozymes are self-cleaving RNA molecules that act as enzymes in RNA processing and ribosome function.
• The genetic code uses triplet codons, is redundant but not ambiguous, to specify amino acids.
• Charged tRNA contains an anticodon end that binds to mRNA and an acceptor end that carries the specific amino acid.
• Ribosomes, made of 30S and 50S subunits, are the site of protein synthesis.
• The 30S subunit (16S rRNA + 21 proteins) recognizes and binds mRNA during initiation.
• The 50S subunit (5S and 23S rRNA + 34 proteins) forms peptide bonds.
• The prokaryotic ribosome (70S) functions as a molecular machine for translation.
• The reading frame is determined by the Shine-Dalgarno sequence, which is upstream of the start codon.
• Chaperone proteins help in folding and prevent misfolding.

Antibiotics

• Rifampicin inhibits bacterial RNA polymerase.
• Streptomycin binds to the 30S ribosomal subunit, disrupting protein synthesis.
• Transcription and translation are coupled in prokaryotes.
• Multiple ribosomes translate a single mRNA at the same time within the polysome, or polyribosome.
• Transcriptional regulation controls when and how much a gene is expressed by controlling the binding of RNA polymerase to DNA.
• Regulatory proteins (transcription factors) bind to specific DNA sequences near promoters, and influence transcription either positively (activators) or negatively (repressors).
• Induction activates gene expression by removing a repressor using an inducer molecule.
• Repression inhibits gene expression by activating a repressor protein with a corepressor, which then blocks transcription. Derepression occurs when the corepressor separates, allowing transcription to resume.
• Activators enhance transcription, often requiring an inducer for DNA binding.
• The lac operon is regulated by LacI repressor (negative regulation) and cAMP-CRP (positive regulation).
• LacI blocks transcription until lactose binds and releases it, and cAMP-CRP enhances transcription under low glucose via increased cAMP levels.
• Low glucose elevates cAMP, which binds CRP to enhance lac operon transcription which functions in diauxic growth.
• Two-component systems allow bacteria to sense external signals and alter gene expression through sensor kinases and response regulators.
• Sigma factors are specialized RNA polymerase subunits that recognize different promoter sequences.
• Alternative sigma factors respond to stress, heat shock, or environmental changes.
• RpoH (σ³²) regulates heat shock genes, and DnaK chaperone proteins refold or degrade damaged proteins, preventing protein aggregation under stress.
• sRNAs regulate gene expression by, inhibiting or activating translation, promoting mRNA degradation, or stabilizing it.
• Riboswitches are mRNA elements that bind metabolites, causing structural changes that affect translation or degradation.
• Quorum sensing allows bacteria to detect population density using autoinducer signaling molecules, leading to altered gene expression for biofilm formation, virulence factor production, or bioluminescence.

Mutations

• Spontaneous mutations include tautomeric shifts (base pairing changes), deamination (loss of amino group), and depurination (loss of a purine base)
• Induced mutations include UV radiation (thymine dimers) and reactive oxygen species (ROS)
• Photoreactivation repairs thymine dimers using light-activated enzymes.
• Base excision repair removes damaged bases and replaces them.
• Mismatch repair detects and corrects replication errors using methylation markers.
• In heavy DNA damage SOS response introduces mutations as a last resort.
• Non-homologous end joining (NHEJ) repairs double-strand breaks but can cause deletions.