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Molecular Biology Notes: DNA, RNA, Replication, Transcription, Translation, and Operons (copy)

DNA Structure and Components

  • DNA is a double helix made of two long polynucleotide chains that run anti-parallel to each other.
  • Each nucleotide contains three parts:
    • Phosphate group
    • Deoxyribose sugar
    • Nitrogenous base (A, T, G, C)
  • The sugar–phosphate backbone is formed by phosphodiester bonds linking the 5' phosphate of one nucleotide to the 3' hydroxyl of the next.
  • Bases pair through hydrogen bonds: A pairs with T (2 bonds) and G pairs with C (3 bonds).
  • The two backbones form a major and a minor groove that regulate protein interactions with DNA.
  • Carbon numbering around deoxyribose: the 1' carbon attaches to the base, 3' and 5' ends define directionality; the 5' end carries a phosphate group while the 3' end has a hydroxyl group.
  • Strands are anti-parallel: one strand runs 5'→3' and the other runs 3'→5'.
  • Chargaff’s rules (for dsDNA): [A] = [T], \, [G] = [C]. In RNA, pairing involves uracil instead of thymine: A = U\,,\ G = C.
  • Conceptual significance:
    • Specific base pairing enables accurate replication and transcription.
    • The chemical asymmetry (5' vs 3') drives directional synthesis of new strands.
    • Hydrogen bonding provides specificity while base stacking contributes to stability.

DNA Packaging: Prokaryotes vs Eukaryotes

  • Prokaryotes
    • DNA is typically circular and resides in the nucleoid; no membrane-bound nucleus.
    • DNA is compacted with small, histone-like proteins (e.g., HU, IHF); supercoiling helps fit DNA into the cell.
    • Often a single origin of replication per circular chromosome; plasmids frequently carry extra genes.
  • Eukaryotes
    • DNA is linear and enclosed within a nucleus; organized into chromatin.
    • DNA wraps around histone octamers to form nucleosomes (H2A, H2B, H3, H4; H1 linker). This forms a 10 nm “beads-on-a-string” and higher-order structures (30 nm fibers, loops).
    • Chromatin exists in euchromatin (more open, transcriptionally active) and heterochromatin (more condensed, repressed).
    • Replication occurs in the nucleus with multiple origins of replication across linear chromosomes.
  • Key contrasts:
    • Packaging complexity: simple in prokaryotes vs highly organized in eukaryotes.
    • Histones: largely absent in prokaryotes; abundant in eukaryotes.
    • Genome organization: circular, fewer distinct chromosome bodies in prokaryotes vs multiple linear chromosomes in eukaryotes.

DNA Replication: Steps and Protein Roles

  • Core sequence of events at a replication origin:
    • Helicase (unwinds the double helix by breaking hydrogen bonds) → creates replication fork.
    • Single-strand binding proteins (SSBs; in bacteria often called SSB) coat exposed single strands to prevent re-annealing.
    • Primase lays down RNA primers to provide a 3' OH for DNA polymerases to begin synthesis.
    • DNA polymerase III (Pol III) extends the new DNA strand in a 5'→3' direction.
    • On the leading strand: synthesis is continuous toward the replication fork.
    • On the lagging strand: synthesis is discontinuous as Okazaki fragments (each requiring a primer).
    • Primers are removed and replaced with DNA by DNA polymerase I.
    • DNA ligase seals nicks between Okazaki fragments to produce a continuous strand.
  • Roles of the named proteins:
    • Helicase: unwinds the helix at the origin to expose single strands.
    • Binding proteins (SSB): stabilize single-stranded DNA and prevent re-annealing.
    • Primase: synthesizes short RNA primers for DNA polymerases to start synthesis.
    • DNA polymerase III: main enzyme that adds nucleotides to the growing DNA strand (high processivity).
    • DNA polymerase I: removes RNA primers and fills in gaps with DNA.
    • Ligase: seals nicks in the sugar-phosphate backbone to finalize the newly synthesized strand.

Directionality and Leading/Lagging Strands

  • DNA polymerase III synthesizes DNA in the 5'→3' direction and reads the template in the 3'→5' direction.
  • Lead strand: synthesized continuously toward the replication fork (toward the opening of the fork).
  • Lagging strand: synthesized discontinuously as short Okazaki fragments away from the fork.
  • Each Okazaki fragment begins with a short RNA primer and is later processed (primer removal + gap filling) and ligated.
  • Practical takeaway: replication is semi-discontinuous, with one continuous strand and one discontinuous strand per replication bubble.

Differences Between RNA Nucleotides and DNA Nucleotides

  • Sugar: RNA has ribose with a hydroxyl at the 2' carbon; DNA has deoxyribose (lacks 2' OH).
  • Bases: RNA uses uracil (U) instead of thymine (T); DNA uses thymine.
  • Structure: RNA is typically single-stranded and can fold into complex structures; DNA is typically double-stranded and forms a stable double helix.
  • Stability and function: DNA is your long-term genetic material; RNA acts as messenger, regulator, and catalytic molecules in various contexts.

Complementary Base Pairing Rules

  • DNA–DNA: A = T,\; G = C (A pairs with T; G pairs with C).
  • DNA–RNA during transcription (template DNA to RNA): A = U,\; G = C; the coding strand of DNA has the same sequence as the mRNA (T replaced by U).
  • Directionality: transcription proceeds 5'→3' on the RNA, using a DNA template read 3'→5'.

Semi-conservative DNA Replication

  • Definition: Each daughter DNA molecule contains one parental (old) strand and one newly synthesized (new) strand.
  • This was demonstrated by the Meselson–Stahl experiment and contrasts with fully conservative or dispersive models.

Transcription: Process and Key Elements

  • Key terms:
    • Template strand (antisense): the DNA strand that is read by RNA polymerase to synthesize RNA.
    • Coding strand (sense): has the same sequence as the RNA (except T instead of U); not used as the template.
    • mRNA strand: the RNA transcript that will be translated into protein.
    • Promoter: DNA sequence where RNA polymerase binds to initiate transcription.
    • Operator: regulatory DNA sequence where repressors can bind to block transcription (characteristic of many prokaryotic operons).
    • Control region: includes promoter and other regulatory sequences that modulate transcription.
  • General steps:
    • Initiation: RNA polymerase binds promoter, DNA opens to form a transcription bubble.
    • Elongation: RNA polymerase synthesizes RNA in the 5'→3' direction, using the template strand.
    • Termination: RNA synthesis ends at a termination signal (rho-dependent or intrinsic). In prokaryotes, transcripts often do not undergo extensive processing; in eukaryotes, processing includes capping, splicing, and polyadenylation.

RNA Splicing: Process and Organisms Involved

  • What is splicing?
    • Removal of non-coding regions (introns) from pre-mRNA and joining of coding regions (exons).
  • Key players:
    • Spliceosome (majority of splicing in eukaryotes): small nuclear ribonucleoproteins (snRNPs) and other factors.
    • Lariat formation via 2' OH of an intron attacking the 5' splice site;
  • Organisms that perform splicing:
    • Eukaryotes (ubiquitous); most introns in higher eukaryotes are removed by spliceosomes.
    • Some archaea and bacteria have self-splicing introns (Group I/II introns) that do not require a spliceosome.

Translation: Process, Components, and Key Concepts

  • Context: Translation occurs on ribosomes in the cytoplasm (bacteria) or cytoplasm of eukaryotes; ribosomes are composed of rRNA and proteins.
  • Key players:
    • mRNA: carries codons that specify amino acids.
    • tRNA: adapts codons to amino acids via anticodons; carries amino acids attached by aminoacyl-tRNA synthetases.
    • Ribosomes: ribosomal subunits (prokaryotes: 50S + 30S; eukaryotes: 60S + 40S) assembling around mRNA.
    • Start codon: typically AUG; initiator tRNA delivers methionine (fMet in bacteria).
    • Stop codons: UAA, UAG, UGA terminate translation.
  • Major steps:
    • Initiation: small ribosomal subunit binds the mRNA near the 5' end; initiator tRNA binds the start codon in the P site; large subunit associates to form the complete ribosome.
    • Elongation: tRNAs enter the A site, peptide bond formation occurs between amino acids in the P and A sites, ribosome translocates to the next codon. A, P, E sites coordinate tRNA movement.
    • Termination: stop codon enters the A site; release factors promote release of the polypeptide and disassembly of the ribosome.
  • Directionality: translation reads mRNA 5'→3'; protein synthesis proceeds amino to carboxyl terminus.

tRNA Structure

  • Classic cloverleaf structure includes:
    • Anticodon loop that recognizes codons on mRNA.
    • Acceptor stem ending in CCA where the amino acid is attached.
    • D arm and TψC arm that stabilize the tRNA’s folded form.
  • In 3D, tRNA adopts an L-shaped structure that positions the amino acid and anticodon appropriately for function.

Practice: Transcription and Translation Example

  • Given template DNA sequence: TACGGATCG
  • Step 1: Transcribe to mRNA (complementary to template, replace T with U):
    • RNA: AUGCCUAGC
  • Step 2: Translate the mRNA sequence using the genetic code:
    • Codons: AUG | CCU | AGC
    • Amino acids: AUG = Met, CCU = Pro, AGC = Ser
    • Resulting peptide: Met-Pro-Ser
  • Important notes:
    • The exact amino acid sequence depends on the reading frame; ensure the correct start site (AUG) is used.
    • If the given sequence was misoriented, a different frame could yield different amino acids.

Constitutive vs Facultative Genes

  • Constitutive genes
    • Continuously expressed under most conditions (housekeeping genes).
    • Example: genes for essential metabolism in rapidly growing cells.
  • Facultative genes
    • Not always expressed; expression is conditional based on environmental cues or cellular needs.
    • Examples: genes for lactose metabolism are induced when lactose is present; many stress response genes are induced under stress.

Operons: Structure and Regulation

  • An operon is a cluster of genes co-transcribed from a single promoter as a polycistronic mRNA.
  • Core components commonly present in operons:
    • Promoter: where RNA polymerase binds to initiate transcription.
    • Operator: binding site for repressor proteins; regulates transcription.
    • Structural genes: coding sequences that encode proteins involved in a common pathway (e.g., lacZ, lacY, lacA in the lac operon).
    • Regulatory gene (sometimes separate): encodes repressor that controls the operon.
  • Regulatory features:
    • A binding site for an activator (e.g., CAP site) can enhance transcription when bound by an activator in response to signals like cAMP levels.
    • Inducers (e.g., allolactose in lac operon) can bind repressors, reducing their affinity for the operator and turning transcription on.
    • Corepressors or end products (e.g., tryptophan in the trp operon) can activate repressors to turn transcription off.

Inducible vs Repressible Operons

  • Inducible operons
    • Usually off but can be turned on by an inducer that inactivates the repressor (e.g., lac operon with allolactose).
  • Repressible operons
    • Usually on but can be turned off when a corepressor (often the end product) binds to the repressor to enable DNA binding (e.g., trp operon).

Escherichia coli luc Operon (Lux System) … Bioluminescence Regulation

  • The lux (luciferase) operon encodes the enzymes required for bacterial bioluminescence (e.g., luxA, luxB for luciferase; luxCDE for substrate synthesis).
  • Regulation typically relies on quorum sensing:
    • LuxI synthesizes an autoinducer (AHL).
    • When autoinducer concentration increases (high cell density), it binds LuxR to form a LuxR–AHL complex.
    • The LuxR–AHL complex activates transcription from the lux promoter (P_lux), increasing expression of lux genes and thereby luminescence.
  • Biochemical reaction (luminescence): luciferase catalyzes the oxidation of a long-chain aldehyde with FMNH2 and oxygen to emit light, producing the observable glow.
  • Practical use: serves as a reporter system for gene expression and promoter activity in E. coli and other hosts.

Escherichia coli trp Operon: Regulation and Attenuation

  • The trp operon is a repressible operon that synthesizes tryptophan biosynthesis enzymes (trpE, trpD, trpC, trpB, trpA).
  • Regulation by TrpR repressor:
    • TrpR repressor binds tryptophan (corepressor) to form an active complex.
    • The TrpR–tryptophan complex binds the operator to block transcription of the operon when tryptophan is abundant.
  • Attenuation mechanism (leader peptide):
    • A leader sequence (trpL) with tryptophan codons is transcribed before the operon.
    • Translated leader peptide influences the formation of terminator or anti-terminator structures in the leader region.
    • High tryptophan: ribosome quickly translates the leader peptide, promoting a terminator hairpin and termination of transcription (attenuation), reducing operon expression.
    • Low tryptophan: ribosome stalls at tryptophan codons in the leader, preventing terminator formation and allowing transcription of the structural genes.

Connections to Foundational Principles and Real-World Relevance

  • Central dogma integration: DNA replication, transcription, and translation are the core processes by which genetic information flows into functional products (proteins/RNA).
  • Regulation of gene expression allows organisms to adapt to environment with energy efficiency; operons provide a compact means to co-regulate functionally related genes.
  • Biotechnological applications include using operon-based promoters for controlled expression, luciferase reporters for monitoring gene activity, and attenuation mechanisms for synthetic biology circuit design.
  • Ethical and practical implications:
    • Understanding gene regulation informs antibiotic target development (e.g., operon regulation in pathogens).
    • Engineered regulatory systems (like lux reporters) enable safe, trackable gene expression in research and industry, with biosafety considerations.

Quick Practice Reference: Key Equations and Rules

  • Base pairing rules (DNA): [A] = [T],\ [G] = [C]
  • RNA pairing rules (transcription context): A = U,\ [G] = [C]
  • Translation reading frame reminder: start at AUG, read in sets of three nucleotides (codons) from 5' to 3' on mRNA.
  • DNA synthesis directionality: polymerases synthesize 5'→3' while reading template 3'→5'.
  • Semi-conservative replication outcome: each daughter DNA contains one old strand and one new strand.

Exam Tip Summary

  • Be able to label a DNA molecule with all components: nucleotides, phosphate groups, sugars, nitrogenous bases, phosphodiester bonds, hydrogen bonds, backbones, 5' and 3' ends, and carbon numbering around deoxyribose.
  • Distinguish prokaryotic vs eukaryotic packaging and explain why histones are central to eukaryotic DNA organization.
  • Sequence the steps of replication and name the correct proteins at each step; explain leading vs lagging synthesis and why primers are needed.
  • Explain why DNA polymerase III works only 5'→3' and how this creates Okazaki fragments on the lagging strand.
  • Differentiate RNA vs DNA nucleotides and base-pairing rules in both DNA–DNA and DNA–RNA contexts.
  • Describe transcription in terms of template/coding strands, promoter, operator, and control region; distinguish prokaryotic vs eukaryotic transcription features.
  • Describe RNA splicing locations and organisms that perform splicing; know the role of spliceosome and introns/exons.
  • Describe translation, including ribosomes, mRNA, tRNA, start/stop codons, and the A/P/E sites, with an emphasis on frame and start signal.
  • Be able to draw and label a tRNA molecule and explain its anticodon–amino acid pairing.
  • Perform a transcription and translation exercise from a given DNA template (including recognizing frame and start codon).
  • Define constitutive vs facultative genes and provide examples.
  • Recognize operon structure (promoter, operator, structural genes, regulatory gene) and how inducers/corepressors modulate transcription.
  • Explain Lac operon, Trp operon, and how attenuation adds another layer of control in the Trp system.
  • Describe the luciferase (lux) operon in E. coli, including the role of quorum sensing and the genes involved (luxA, luxB, luxCDE) and how luminescence is produced.
  • Understand how the TrpR repressor and attenuation work together to regulate the Trp operon and why this is advantageous for the cell.