Key promoter elements in E. coli:

1. -10 Region:

   • Consensus sequence: 5'-TATAAT-3'
   • Located ~10 bp upstream of transcription start site
   • Function: Site of DNA melting during open complex formation
   • Bound by σ factor of RNA polymerase

2. -35 Region:

   • Consensus sequence: 5'-TTGACA-3'
   • Located ~35 bp upstream of transcription start site
   • Function: Initial recognition site for RNA polymerase binding
   • Critical for polymerase recruitment

3. UP Element:

   • AT-rich sequence located between -40 and -60
   • Not present in all promoters
   • Function: Enhances transcription when present
   • Bound by α subunits of RNA polymerase

4. Spacer regions:

   • Between -10 and -35 regions (optimal spacing: 17 nucleotides)
   • Between -10 region and transcription start site (6-8 nucleotides)
   • Proper spacing is critical for promoter strength and efficiency

Promoter strength correlates with how closely these elements match their consensus sequences.

Ribozyme-Catalyzed Splicing: • First discovered in group I introns of Tetrahymena thermophila rRNA • RNA molecules catalyze their own splicing without protein enzymes • Uses transesterification reactions that maintain phosphodiester bond energy balance • No high-energy cofactors (like ATP) required for the reaction • In group I introns: guanosine 3'-OH acts as nucleophile in first reaction step • In group II introns: 2'-OH of internal A residue acts as nucleophile

Revolutionary Impact: • Overturned the central dogma that only proteins could serve as biological catalysts • Provided evidence for the "RNA world" hypothesis of early evolution • Suggested RNA could have preceded proteins in early life forms • Demonstrated that RNA can have both informational and catalytic functions • Expanded understanding of RNA beyond just information transfer • Led to discovery of other ribozymes (ribosomal RNA, RNase P) • Earned Thomas Cech and Sidney Altman the 1989 Nobel Prize in Chemistry

This discovery fundamentally changed our view of RNA from a passive carrier of information to an active participant in cellular biochemistry and provided insight into the possible origins of life.

Eukaryotic primary transcripts undergo three essential processing steps:

1. 5' capping - Addition of a 7-methylguanosine cap linked by an unusual 5',5'-triphosphate bond to the 5' end, which protects the mRNA from degradation and aids in ribosome binding

2. Splicing - Removal of non-coding introns and joining of coding exons to create a continuous coding sequence

3. 3' end processing - Cleavage of the 3' end followed by addition of a poly(A) tail (typically 80-250 adenine nucleotides)

All these processes occur in the nucleus before the mature mRNA is exported to the cytoplasm.

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.

Reaction catalyzed: RNA + nATP → RNA-(AMP)n + nPPi where n = 80 to 250

Key differences from typical nucleic acid polymerization:

• Template-independent: Does not require a DNA or RNA template to specify the sequence
• Adds only adenosine (A) residues regardless of input
• Requires a cleaved RNA primer with a free 3'-OH group
• Creates a defined sequence (all A's) based solely on enzyme binding site architecture
• Continues until the appropriate tail length is achieved (regulated by associated proteins)

This represents a rare example of a nucleic acid synthesis reaction where sequence information is encoded in the enzyme itself rather than in a template.

• A hammerhead ribozyme is a small RNA enzyme (~41 nucleotides) that catalyzes site-specific RNA cleavage

• Biological role:

  • Found in virusoids (virus-like elements with small RNA genomes)
  • Involved in self-cleaving reactions during replication
  • Helps virusoids, which typically need assistance from another virus for replication/packaging

• Minimal structural requirements:

  • Three helical stems arranged in a hammer-like shape
  • Conserved central catalytic core with specific nucleotides (especially the conserved nucleotides CUGA)
  • Specific stem-loop structure that brings the cleavage site into proper orientation
  • Requires Mg²⁺ ions for catalytic activity (metalloenzyme function)

• Cleavage occurs at a specific phosphodiester bond positioned within the conserved core

• Demonstration of RNA enzymatic function:

  • Catalyzes reactions without protein components
  • Shows defined substrate specificity
  • Adopts specific 3D structure necessary for function
  • Exhibits accelerated reaction rates compared to uncatalyzed reactions
  • Functions as a true metalloenzyme (requires Mg²⁺)

• Chemical reaction catalyzed:

  • Site-specific phosphodiester bond hydrolysis (RNA cleavage)
  • Specifically targets the phosphodiester bond following a conserved sequence
  • Creates 5'-hydroxyl and 2',3'-cyclic phosphate termini
  • Reaction mechanism involves in-line nucleophilic attack by the 2'-OH on the phosphorus atom
  • Self-cleavage occurs without requiring external energy sources (ATP)

• This ribozyme provides evidence for the "RNA world" hypothesis, suggesting RNA molecules could serve both genetic and catalytic roles in early evolution

SELEX (Systematic Evolution of Ligands by Exponential enrichment):

Key steps:

1. Begin with a random pool of RNA sequences (~10^15 different molecules)
2. Apply selection pressure by passing through a column with target molecule attached
3. Collect RNA molecules that bind the target
4. Amplify selected RNAs (RT-PCR then transcription)
5. Repeat selection-amplification cycle 12+ times to enrich high-affinity binders

Limitations:

• Practical complexity limit of ~10^15 sequences (complete randomization limited to 25 nucleotides)
• When using longer RNAs, only a fraction of possible sequences can be screened
• Selection based only on binding, additional screening needed for catalytic function
• May select for non-specific binding or column artifacts

Applications: Generating aptamers for therapeutic targets, identifying catalytic RNAs, developing detection reagents.

Critical experimental criteria:

1. Demonstrating prebiotic synthesis of RNA building blocks
   • Successfully shown for purines like adenine from simple precursors (HCN)
2. Demonstrating RNA's catalytic capacity
   • Natural ribozymes (rRNA introns, RNase P)
   • Artificially selected ribozymes via SELEX showing diverse activities:
     • Ester/amide bond formation
     • Carbon-carbon bond formation
     • Metallation of porphyrins
     • SN2 reactions

Key gaps to address:

1. Self-replication - Creating an RNA that can fully replicate itself
2. Metabolic pathways - Demonstrating RNA catalysis of precursor synthesis
3. Energy utilization - Explaining how early systems captured/used energy
4. Transition to DNA/protein world - Mechanisms for evolution to modern systems
5. Compartmentalization - How primitive cells might have formed
6. Prebiotic plausibility - Realistic conditions for sufficient RNA synthesis

Each criterion represents a testable aspect of the hypothesis, with ongoing research addressing these questions.

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.