RNA Metabolism

• Nucleotide addition mechanism:

  1. Incoming NTP base-pairs with template DNA base
  2. 3'-OH of RNA attacks α-phosphate of incoming NTP
  3. Pyrophosphate (PPi) is released
  4. RNA strand extends by one nucleotide
  5. Polymerase translocates to next template position

• Role of metal ions:

  • Two Mg²⁺ ions are essential for catalysis
  • First Mg²⁺: Facilitates attack by 3'-OH on α-phosphate
  • Second Mg²⁺: Facilitates leaving of pyrophosphate
  • Both ions stabilize the pentacovalent transition state
  • Ions are coordinated by three conserved Asp residues (460, 462, 464 in E. coli)

Note: This two-metal-ion catalysis mechanism is conserved among all nucleic acid polymerases, highlighting its evolutionary importance.

• An ~8 base pair region where newly synthesized RNA temporarily pairs with the DNA template strand
• Key functions:
  • Stabilizes the transcription complex during elongation
  • Maintains the register of transcription (keeping RNA polymerase on track)
  • Contributes to transcription fidelity by providing a proofreading checkpoint
  • Enables the transient separation of DNA strands
• Dynamic properties:
  • Constantly forms at the 3' end of the growing RNA chain
  • Constantly dissociates at its upstream end as the DNA duplex reforms
  • Helps guide the RNA out of the polymerase complex
  • Maintains the correct reading frame during rapid synthesis (~50-90 nucleotides/second)
• Critical for preventing premature termination of transcription

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.

Structure: • Long tail consisting of multiple repeats of consensus heptapeptide sequence YSPTSPS • 27 repeats in yeast (18 exact matches to consensus) • 52 repeats in mice/humans (21 exact matches) • Separated from main enzyme body by unstructured linker sequence

Functions: • Undergoes extensive phosphorylation during transcription initiation and elongation • Phosphorylation causes conformational change in the complex to initiate transcription • Serves as binding platform for RNA processing factors (capping, splicing, polyadenylation) • Coordinates coupling between transcription and post-transcriptional processing • Acts as assembly point for protein complexes involved in mRNA maturation • Helps recruit nucleotide-excision repair proteins to damaged sites in actively transcribed genes

The phosphorylation state of the CTD changes throughout the transcription cycle, allowing it to interact with different factors at appropriate times.

Key structural features of actinomycin D:

1. Planar phenoxazone ring system (the orange-shaded portion):

   • Intercalates between successive G≡C base pairs in DNA
   • Creates physical disruption in DNA structure

2. Two cyclic peptide structures (containing Sar, L-Pro, L-meVal, D-Val, L-Thr):

   • Bind specifically to the minor groove of DNA
   • Stabilize the drug-DNA complex
   • Enhance specificity for certain DNA sequences

Mechanism of action:

• The intercalation plus minor groove binding creates a stable complex
• This complex physically blocks RNA polymerase progression
• DNA develops a bend at the intercalation site
• The polymerase cannot move past this structural distortion
• Transcription elongation halts while the drug remains bound
• This mechanism allows actinomycin D to function at low concentrations without disrupting DNA replication

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. Initial assembly:
   • U1 snRNP binds 5' splice site via base pairing
   • U2 snRNP binds branch site, creating bulge that exposes adenosine nucleophile
2. Conversion to inactive spliceosome:
   • U4/U6·U5 tri-snRNP complex joins (ATP-dependent)
3. Activation step:
   • U1 and U4 are expelled
   • U6 rearranges to pair with 5' splice site and with U2
   • Forms catalytically active structure
4. Catalytic steps follow group II intron chemistry pattern
   • First transesterification: lariat formation
   • Second transesterification: exon joining and intron release

• Ribozyme defining properties: • RNA molecule that catalyzes specific chemical reactions • Has defined active site and catalytic core • Shows substrate specificity • Can accelerate reactions by similar mechanisms as protein enzymes • Often requires metal ions (typically Mg²⁺) as cofactors

• Hammerhead ribozyme specifics: • Minimal catalytic structure with only 41 nucleotides • Contains highly conserved core nucleotides essential for function • Catalyzes site-specific RNA self-cleavage via transesterification • Forms precise three-dimensional structure with defined active site • Requires Mg²⁺ ions for catalysis (metalloenzyme) • Can function in trans (with separate substrate strand)

• L-19 IVS is a 395-nucleotide RNA derived from the Tetrahymena intron that catalyzes nucleotidyl transfer reactions
• Key parameters:
  • Processes ~100 substrate molecules per hour
  • Shows Michaelis-Menten kinetics
  • kcat/Km (specificity constant) = 10³ M⁻¹s⁻¹
  • Accelerates hydrolysis by a factor of 10¹⁰ compared to uncatalyzed reaction
• Uses three catalytic strategies:
  • Substrate orientation
  • Covalent catalysis
  • Metal-ion catalysis
• Best substrates are oligonucleotides (like (C)₅) that can base-pair with the guanylate-rich internal guide sequence

Group I and II introns represent two distinct self-splicing mechanisms:

Group I Intron Splicing

1. First step nucleophile: 3' OH of an external guanosine molecule
2. Reaction pathway:
   • Guanosine attacks the 5' splice site
   • Forms new phosphodiester bond between G and 5' end of intron
   • Produces linear excised intron
3. Second step: 3' OH of 5' exon attacks 3' splice site, joining exons

Group II Intron Splicing

1. First step nucleophile: 2' OH of a specific internal adenosine within the intron
2. Reaction pathway:
   • Adenosine attacks the 5' splice site
   • Forms distinctive 2',5'-phosphodiester bond
   • Creates characteristic lariat intermediate (A with 3 phosphodiester bonds)
3. Second step: 3' OH of 5' exon attacks 3' splice site, joining exons

Key Similarities

• Both use transesterification reactions (ATP-independent)
• Both maintain the same number of phosphodiester bonds
• Both exhibit ribozyme (RNA catalytic) activity
• Both use the 3' OH of the 5' exon in their second step

Key Differences

• Different first-step nucleophiles (external G vs. internal A)
• Different intermediates (linear vs. lariat)
• Group II mechanism closely resembles nuclear pre-mRNA splicing by the spliceosome