Molecular Information Flow & Protein Processing – Comprehensive Bullet-Point Notes

6.1 DNA and Genetic Information Flow

• Gene as functional unit
  • Encodes heritable information; illustrated in Figure 6.1a.
  • Genes reside on larger genetic elements (chromosomes, plasmids, viral genomes, etc.).
• Genome = total collection of genetic elements in a cell/virus.
• Informational macromolecules
  • DNA (genetic blueprint)
  • RNA (transcription product)
  • Protein (translation product of mRNA)
• Nucleotides (monomers of nucleic acids)
  • Three parts: pentose sugar (ribose in RNA, deoxyribose in DNA), nitrogenous base, phosphate.
  • Without phosphate = nucleoside.
• Nitrogenous bases
  • Purines: adenine (A), guanine (G) – occur in both DNA & RNA.
  • Pyrimidines: cytosine (C) in DNA/RNA; thymine (T) in DNA; uracil (U) in RNA.
• Double-helix properties
  • Sugar-phosphate backbone held by phosphodiester bonds (link 3′-C of one sugar to 5′-C of next).
  • Complementary base pairing: A–T (or A–U in RNA), G–C.
  • Strands are antiparallel; major and minor grooves (proteins typically bind the major groove).
• DNA size & supercoiling
  • Size expressed in base pairs: $1000\,\text{bp}=1\,\text{kbp}$; $10^{6}\,\text{bp}=1\,\text{Mbp}$.
  • E. coli genome ≈ $4.64\,\text{Mbp}$.
  • Linear DNA length >> cell size ⇒ negative supercoiling compacts it.
  • Topoisomerases add/remove supercoils.
  • DNA gyrase inserts negative supercoils via double-strand breaks.
  • Positive supercoiling (in some thermophilic Archaea) resists heat-induced strand separation.
• Central Dogma & gene expression (Fig 6.5)
  • Replication $\rightarrow$ transcription $\rightarrow$ translation.
  • Main RNA classes: mRNA (information carrier), tRNA (adaptor), rRNA (catalytic/structural).
  • Replication by DNA polymerase; transcription by RNA polymerase; translation on ribosomes.
• Eukaryote vs. Prokaryote information flow
  • Eukaryotes: monocistronic mRNAs; replication/transcription in nucleus; translation in cytoplasm.
  • Prokaryotes: often polycistronic mRNAs; transcription & translation are coupled (Fig 6.6).
  • Some viruses violate the central dogma (e.g., RNA-to-DNA via reverse transcriptase).

6.2 Genetic Elements: Chromosomes and Plasmids

• Chromosome = primary genetic element; usually single circular in Bacteria/Archaea, multiple linear in Eukarya.
• Other elements (Table 6.1)
  • Viral genomes (DNA or RNA, ss or ds, circular/linear).
  • Plasmids (dsDNA, extrachromosomal, circular/linear, self-replicating).
  • Organellar genomes (mitochondria, chloroplasts).
  • Transposable elements (always inserted in another DNA molecule).
• Plasmid features
  • Typically <5 % of chromosome size; copy number ranges 1–100+.
  • Non-essential but may confer advantageous traits: antibiotic resistance (R-plasmids, e.g., R100), virulence factors, bacteriocins, nitrogen fixation, hydrocarbon degradation.
• Transposable elements
  • Mobile DNA segments that move within/between DNA molecules (chromosome, plasmid, virus) in both prokaryotes & eukaryotes.
• E. coli K-12 chromosome (Fig 6.7)
  • ~5 Mbp, ~4300 protein-coding genes (88 % coding).
  • Operons cluster some pathway genes, but many pathways are dispersed – operons are exceptions, not the rule.

6.3 Templates, Enzymes, and the Replication Fork

• Semiconservative replication (Fig 6.10)
  • Each daughter duplex: one parental + one new strand.
• DNA polymerases in E. coli (Table 6.2)
  • DNA Pol III = main chromosomal replicase.
  • DNA Pol I removes RNA primers & fills gaps.
  • Others function in repair.
• Primer requirement
  • DNA polymerase adds nucleotides to pre-existing 3′-OH; primer synthesized by primase (RNA) (Fig 6.11).
• Initiation at oriC
  • DnaA binds origin $\rightarrow$ opens duplex.
  • Helicase (DnaB) + loader (DnaC) unwind DNA; primase lays down RNA primer; polymerases assemble.
• Leading vs. Lagging strands (Figs 6.13, 6.14)
  • Leading: continuous 5′$\rightarrow$3′ synthesis toward fork.
  • Lagging: discontinuous synthesis away from fork; multiple primers form Okazaki fragments.
  • DNA Pol I replaces RNA primers; DNA ligase seals nicks (requires ATP or NADH).

6.4 Bidirectional Replication, the Replisome, and Proofreading

• Bidirectional θ-mode replication (Fig 6.15) in circular chromosomes: two forks move opposite directions.
• Replisome (Fig 6.16)
  • Multi-protein complex: primosome (helicase + primase), 2 DNA Pol III cores, tau dimerization subunit holds cores, sliding clamps, clamp loader, SSB, DNA gyrase, etc.
• Speed & fidelity
  • DNA Pol III ≈ $1000\,\text{nt s}^{-1}$.
  • Proofreading 3′$\rightarrow$5′ exonuclease reduces error rate to $10^{-8}!\text{–}!10^{-11}$ per bp (Fig 6.17).

6.5 Transcription in Bacteria

• Chemical distinctions RNA vs. DNA
  • Ribose sugar; uracil replaces thymine; usually single-stranded; can form secondary structures.
• RNA polymerase
  • Holoenzyme = core (α₂ββ′ω) + σ factor (Fig 6.19).
  • σ recognizes promoter consensus sequences: –10 Pribnow box (TATAAT) & –35 region (TTGACA) (Fig 6.20).
  • Alternative σ factors (Table 6.3) regulate stress responses, motility, heat-shock, etc.
• Transcription cycle (Fig 6.18)
  1. σ binds promoter $\rightarrow$ initiation.
  2. σ released; elongation proceeds (≈ 45 nt s⁻¹).
  3. Termination: intrinsic (stem-loop + poly-U, Fig 6.23) or Rho-dependent.
• Transcriptional units & operons
  • Can be monocistronic or polycistronic (Fig 6.22).
  • rRNA operon example: 16S–23S–5S plus tRNA (Fig 6.21).

6.6 Transcription in Archaea and Eukarya

• Polymerases
  • Archaea: single RNA polymerase resembling eukaryotic Pol II.
  • Eukarya: three nuclear polymerases (I–III).
• Promoters
  • Core elements: BRE (B recognition element) + TATA box (6–8 bp) ~18–27 nt upstream; bound by TBP & TFB (Fig 6.24).
• Termination in Archaea
  • Some intrinsic (inverted repeats + AT-rich), some protein-mediated (Eta factor).
• RNA processing
  • Eukaryotes: 5′ cap (m⁷G), 3′ poly(A) tail, splicing via spliceosome (Fig 6.25), then export (Fig 6.26).
  • Archaea: occasional introns in tRNA/rRNA removed by specific endonucleases.

6.7 Amino Acids, Polypeptides, and Proteins

• Protein roles
  • Catalysis (enzymes), structure (membranes, ribosomes), regulation (DNA-binding proteins), etc.
• Amino acid chemistry
  • General formula: $\text{H}_2\text{N}–\text{CH(R)}–\text{COOH}$.
  • Peptide bond formation releases $\text{H}_2\text{O}$ (Fig 6.28).
• Structural levels
  • Primary: linear sequence.
  • Secondary: α-helix & β-sheet via H-bonds (Fig 6.29).
  • Tertiary: 3-D folding (hydrophobic, ionic, S–S) (Fig 6.30).
  • Quaternary: multi-subunit association.
  • Denaturation = loss of structure/function.

6.8 Transfer RNA (tRNA)

• Function: adaptor that matches codon (mRNA) to amino acid.
• Structure (Fig 6.31)
  • ~75–95 nt; cloverleaf secondary; acceptor stem 3′-CCA; anticodon loop.
  • Modified bases (pseudouridine, inosine, etc.).
• Charging by aminoacyl-tRNA synthetases (Fig 6.32)
  1. Amino acid + ATP $\rightarrow$ aminoacyl-AMP + PPᵢ.
  2. Aminoacyl group transferred to 3′ A of tRNA (ester bond) $\rightarrow$ charged tRNA.
  • Recognition ensures fidelity; each aa has its own synthetase.

6.9 Translation and the Genetic Code

• Code basics
  • Triplet codons; 64 possibilities (Table 6.4).
  • Start codon AUG (codes fMet in Bacteria, Met in Archaea/Eukarya).
  • Stop codons: UAA, UAG, UGA.
• Degeneracy & wobble (Fig 6.33)
  • Third-base wobble allows one tRNA to pair with multiple synonymous codons.
  • Codon bias correlates with tRNA abundance.
• Reading frame & RBS
  • Correct frame established by Shine–Dalgarno sequence pairing with 16S rRNA.
  • ORF: AUG → … → stop.

6.10 Mechanism of Protein Synthesis

• Components: mRNA, charged tRNAs, ribosomes (30S + 50S = 70S), translation factors, GTP.
• Initiation (Fig 6.36)
  • 30S binds mRNA at RBS.
  • fMet-tRNA + IF1/IF2/IF3 form initiation complex.
  • 50S joins $\rightarrow$ 70S ready for elongation.
• Elongation (Fig 6.37)
  1. EF-Tu–GTP delivers charged tRNA to A site.
  2. Peptidyl transferase (23S rRNA) forms peptide bond; chain transferred to A-site tRNA.
  3. EF-G–GTP drives translocation: ribosome moves 1 codon; empty tRNA → E site → exit.
• Termination & recycling
  • Stop codon encountered; release factors (RF1/RF2/RF3) hydrolyze peptidyl-tRNA bond.
  • Ribosome dissociates; subunits reused.
• Polysomes (Fig 6.38): many ribosomes translating one mRNA increase protein output.
• Role of rRNA
  • 16S aligns mRNA, 23S catalyzes peptide bond, rRNAs mediate translocation and subunit association.
• Trans-translation (Fig 6.39)
  • tmRNA (tRNA + mRNA hybrid) rescues ribosomes stalled on mRNAs lacking stop codon; tags incomplete polypeptides for degradation.

6.11 Assisted Protein Folding and Chaperones

• Major bacterial chaperones (Fig 6.40a)
  • DnaK/DnaJ: bind nascent chains, ATP-dependent.
  • GroEL/GroES: barrel-type complex provides enclosed folding chamber.
• Heat- & cold-shock response
  • Chaperones refold denatured proteins before proteolysis.
  • Cold-shock proteins prevent RNA secondary structure at low T°.
• Cofactor insertion example (Fig 6.40b)
  • NarJ chaperone inserts molybdenum cofactor into nitrate reductase (NarGH) prior to membrane localization.

6.12 Protein Secretion: The Sec and Tat Systems

• Why secrete?
  • Export enzymes, toxins, surface/outer-membrane proteins, environmental polymers (e.g., halomucin in Haloquadratum walsbyi, Fig 6.41).
• Signal sequence (15–20 aa)
  • N-terminal, +ve residues → hydrophobic core → polar tail.
  • Keeps protein unfolded; directs to translocase (Fig 6.42).
• Sec pathway
  • SecA drives post-translational export of unfolded proteins using ATP.
  • SRP guides membrane proteins co-translationally.
• Tat pathway
  • Twin-arginine motif (RR) in signal; exports folded cofactor-containing proteins.
  • TatBC receptor binds; TatA forms channel.

6.13 Protein Secretion: Gram-Negative Systems

• Overview (Fig 6.43)
  • Types I–VI (and others) form multicomponent channels crossing one or both membranes.
• Two-step systems (require Sec/Tat first)
  • Type II: periplasm → exterior; dedicated secreton pore.
  • Type V: autotransporters; C-terminal β-barrel inserts into outer membrane, external passenger domain secreted by own pore.
• One-step systems (Sec/Tat-independent)
  • Type I: ABC transporter + membrane fusion protein + outer-membrane pore; ATP-driven.
  • Type III: injectisome “needle” injects effector proteins into eukaryotic cells (Fig 6.44a).
  • Type IV: pilus-like, commonly transfers DNA (conjugation) or proteins; most widespread.
  • Type VI: contractile phage-like structure injects toxins into target cells (Fig 6.44b).
• Functions: symbiosis, biofilm formation, pathogenesis, DNA uptake, competition (bacteriocins), antibiotic export.