Viral genome organization demonstrates evolutionary adaptations through:

• Bidirectional coding: Using both DNA strands as templates for transcription, doubling the coding capacity • Overlapping reading frames: The same DNA sequence can encode different proteins depending on where translation starts • Compact regulatory elements: Minimal promoter and terminator regions • Polycistronic messages: Multiple genes expressed from a single transcript (36,000 bp adenovirus genome produces a 25,000 nucleotide primary transcript) • Post-transcriptional processing: Single long transcripts are processed into multiple functional mRNAs

These adaptations allow viruses to maintain small genome sizes (adenovirus: 36,000 bp) while encoding all necessary proteins for infection, replication, and virion assembly—a crucial advantage given space constraints within viral capsids.

Structural Transitions of RNA Polymerase:

Closed Complex:

• σ subunit is bound to core polymerase
• DNA is double-stranded and intact
• Polymerase makes specific contacts with -35 and -10 promoter regions
• No RNA synthesis occurs

Open Complex:

• DNA unwinding creates a transcription bubble (12-15 bp)
• Template strand is positioned at the active site
• Active site becomes accessible to incoming nucleotides
• DNA remains relatively straight through the complex

Elongation Form:

• σ subunit has dissociated from the complex
• RNA-DNA hybrid of ~8 bp forms within the transcription bubble
• Nascent RNA exits through a dedicated channel
• DNA follows a more circuitous path through the complex
• Active site is located in a cleft between β and β' subunits
• Catalytic Mg²⁺ ion is positioned for nucleotide addition

Functional Consequences:

• Processivity dramatically increases
• Stability of the complex increases (less prone to dissociation)
• Nucleotide addition becomes highly efficient and directional
• Complex becomes capable of transcribing thousands of nucleotides without dissociating

DNase I concentration must be carefully optimized to achieve "single-hit kinetics" where:

• Each DNA molecule is cut only once on average
• Too much cutting: destroys all fragments, eliminating the footprint
• Too little cutting: insufficient fragment diversity for analysis

Critical because:

• Multiple cuts per molecule would eliminate the relationship between fragment length and binding site location
• Single cuts ensure the missing bands directly correspond to protected regions
• Proper concentration maintains statistical distribution of fragments covering the entire sequence

This controlled partial digestion creates a ladder of fragments that precisely maps the boundaries of protein-DNA interactions.

Sequential Assembly:

1. TBP (or TFIID) binds to TATA box
2. TFIIB binds to TBP and DNA on either side
3. TFIIA (optional) stabilizes the TFIIB-TBP complex
4. TFIIF-Pol II complex binds to TFIIB-TBP
5. TFIIE binds
6. TFIIH binds, completing the closed complex

Functions of Factors: • TBP: Specifically recognizes TATA box (38 kDa) • TFIIA: Stabilizes binding of TFIIB and TBP at promoter (3 subunits: 12, 19, 35 kDa) • TFIIB: Binds TBP and recruits Pol II-TFIIF complex (35 kDa) • TFIIF: Binds tightly to Pol II; prevents nonspecific DNA binding (2 subunits: 30, 74 kDa) • TFIIE: Recruits TFIIH; has ATPase and helicase activities (2 subunits: 34, 57 kDa) • TFIIH: Unwinds DNA at promoter; phosphorylates Pol II CTD; recruits repair proteins (12 subunits: 35-89 kDa)

The complete assembly has more than 30 polypeptides, not counting TFIIA.

Transesterification in RNA splicing differs from hydrolysis in several key ways:

• In transesterification, a hydroxyl group (like the 3'-OH of guanosine) directly attacks a phosphodiester bond, transferring the phosphate to create a new ester bond
• No water molecule is used as a reactant (unlike hydrolysis which uses H₂O)
• The reaction maintains energy balance (does not require ATP) because a new bond forms as another breaks
• The number of phosphodiester bonds remains constant throughout the reaction
• The reaction is potentially reversible under similar conditions

In contrast, hydrolysis uses water to break bonds, typically requires energy input, and results in bond cleavage without simultaneous bond formation.

The 3'-OH group of guanosine functions as a nucleophile in RNA splicing due to several key properties:

• The hydroxyl group contains an oxygen atom with lone pairs of electrons
• In the correct pH environment (typically neutral to slightly basic), the 3'-OH can be partially deprotonated, increasing its nucleophilicity
• The ribozyme's active site positions the 3'-OH optimally for attack on the phosphodiester bond
• Metal ions (often Mg²⁺) within the active site help coordinate the reaction by:
  • Stabilizing the developing negative charge in the transition state
  • Activating the 3'-OH by lowering its pKa
  • Properly orienting the attacking group and target phosphate

This nucleophilic attack results in the formation of a new phosphodiester bond between guanosine and the 5' end of the intron, while simultaneously breaking the bond between the exon and intron.

1. Recognition and binding: An enzyme complex binds to the cleavage signal sequence containing the conserved AAUAAA sequence located 10-30 nucleotides upstream of the cleavage site

2. Cleavage: An endonuclease in the enzyme complex cleaves the RNA at a site 10-30 nucleotides downstream of the AAUAAA sequence, generating a free 3'-hydroxyl group

3. Polyadenylation: Polyadenylate polymerase adds 80-250 adenosine residues (poly(A) tail) to the 3' end using ATP as substrate, without requiring a template

Note: The poly(A) tail serves as a binding site for proteins that protect the mRNA from degradation, unlike bacterial poly(A) tails which stimulate decay.

The catalytic activity of L-19 IVS has significant evolutionary implications:

• It demonstrates that RNA can function as both a genetic material and a catalyst (ribozyme)
• The ability to perform nucleotidyl transfer reactions shows RNA can modify other RNA molecules
• The cycle demonstrates primitive RNA polymerization capability without protein enzymes
• (C)5 is a preferred substrate because it can base-pair with the guide sequence, showing sequence-specific recognition similar to modern enzymes
• The ribozyme exhibits enzyme-like properties: it follows Michaelis-Menten kinetics, accelerates reactions by a factor of 10^10, and employs substrate orientation, covalent catalysis, and metal-ion catalysis

This supports the "RNA World" hypothesis that RNA-based life forms could have existed before the evolution of DNA and proteins, providing a plausible pathway for the development of more complex biological systems.

Shared Mechanisms & Features

• All three elements utilize reverse transcriptase to convert RNA → DNA
• All integrate DNA copies into genomes
• All likely evolved from common ancestral genetic elements
• Represent a "backwards" information flow contrary to the original Central Dogma

Evolutionary Hierarchy & Distinctions

1. Retrohoming Group II Introns:

   • Most primitive mechanism
   • Found in bacteria and organellar genomes
   • Encode protein with reverse transcriptase activity
   • Direct RNA insertion into DNA followed by reverse transcription

2. Retrotransposons:

   • "Trapped" retroviruses lacking env genes
   • Cannot form viral particles or exit cells
   • Examples: Ty element (yeast), copia element (Drosophila)
   • Comprise up to 45% of the human genome

3. Retroviruses:

   • Most evolved form with complete viral particles
   • Contain gag, pol (encoding RT), and env genes
   • LTRs (Long Terminal Repeats) at both ends
   • Can move between cells through env-mediated exit/entry
   • Evolved from retrotransposons by acquiring env genes

Evolutionary & Genomic Significance

• Ancient Origins: Reverse transcriptase predates multicellular organisms
• RNA World Support: Suggests RNA-based life forms preceded DNA-based organisms
• Genome Complexity: These "selfish" or "parasitic" elements contributed to:
  • Gene duplication
  • Exon shuffling
  • Regulatory element dispersion
  • Chromosomal rearrangements

Note: This evolutionary relationship challenges our view of "junk DNA" by showing that mobile genetic elements were likely fundamental architects of genomic complexity and evolutionary innovation.

Splicing Mechanisms Comparison

Group I Introns

• Nucleophile: External guanosine (G, GMP, GDP, GTP) serves as nucleophile
• First reaction: 3'-OH of guanosine attacks 5' splice site
• Second reaction: 3'-OH of 5' exon attacks 3' splice site
• Intermediate: Forms linear excised intron (no lariat structure)
• Catalysis: Self-splicing (no proteins required in vitro)
• Location: Some nuclear rRNAs, mitochondrial and chloroplast genes
• Mobility: Mobile via homing (DNA-based process)

Group II Introns

• Nucleophile: Internal adenosine 2'-OH within the intron
• First reaction: 2'-OH of branch-site adenosine attacks 5' splice site
• Second reaction: 3'-OH of 5' exon attacks 3' splice site
• Intermediate: Forms branched "lariat" with 2',5'-phosphodiester bond
• Catalysis: Self-splicing (though proteins assist in vivo)
• Location: Mitochondrial/chloroplast mRNAs in fungi, algae, plants, some bacteria
• Mobility: Mobile via retrohoming (RNA intermediate)

Spliceosomal Introns

• Nucleophile: Internal adenosine 2'-OH (same as Group II)
• Mechanism: Same lariat-forming chemistry as Group II introns
• Catalysis: Requires spliceosome complex (U1, U2, U4, U5, U6 snRNPs + ~50 proteins)
• Sequence markers: Typically GU at 5' end and AG at 3' end
• Energy: Requires ATP for spliceosome assembly (not for actual splicing chemistry)
• Location: Eukaryotic nuclear genes

Common Features & Evolutionary Relationships

• All use transesterification reactions (no ATP needed for actual chemistry)
• All maintain phosphodiester bond energy balance
• Group II and spliceosomal introns share mechanistic similarity (lariat formation)
• Spliceosome likely evolved from Group II introns (RNA components provide catalytic activity)
• snRNPs in spliceosome replaced catalytic domains of ancestral Group II introns
• The RNA-based catalytic mechanisms support the RNA world hypothesis

Note: While Group I and Group II introns can self-splice in vitro, in vivo splicing typically involves protein factors that enhance efficiency and specificity.