DNA Transcription and RNA Processing

DNA-Directed RNA Synthesis (Transcription)

Overview

• Synthesis of RNA from DNA. Occurs in cytoplasm (prokaryotes) or nucleus (eukaryotes).

Chemistry

• Direction: $5'$ to $3'$. Nucleophilic attack of $3'-OH$ on $rNTP$ alpha-phosphate results in $PP_i$ release.

• Template (Antisense): Read in $3' → 5'$ direction.

• Coding (Sense): Identical to RNA transcript (but uses $T$ instead of $U$).

RNA Polymerase (RNAP)

• Prokaryotes: Holoenzyme ($\alpha_2\beta\beta'ωσ$). The $\sigma$ factor is essential for promoter recognition ($-10$ and $-35$).

• Eukaryotes:

  • Pol I: Large $rRNA$.

  • Pol II: mRNA, snRNA, miRNAs.

  • Pol III: $tRNA$, $5S$ $rRNA$.

  • Requires General Transcription Factors (GTFs) to form a Pre-Initiation Complex.

Stages

1. Initiation: Promoter binding and DNA unwinding ($Closed → Open$ complex).

2. Elongation: RNAP moves along DNA; transcription bubble maintains an $8–9$ bp DNA-RNA hybrid.

3. Termination:

   • Prokaryotes: Rho-independent (hairpin loop) or Rho-dependent ($ρ$ protein).

   • Eukaryotes: Coupled with polyadenylation signal sequence ($AAUAAA$).

Processing (Eukaryotes)

• $5'$ Capping: $7$-methylguanosine addition for protection.

• $3'$ Polyadenylation: Poly-A tail for stability and export.

• Splicing: Spliceosomes remove introns and join exons.

Translation (RNA-Directed Protein Synthesis)

Overview

• Conversion of mRNA nucleotide sequence into amino acids. Occurs on ribosomes.

Key Components

• tRNA: Adapter molecule with an anticodon and a specific amino acid.

• Aminoacyl-tRNA Synthetase: "Charges" tRNA with its correct amino acid.

• Ribosome Sites:

  • A (Aminoacyl): Incoming tRNA.

  • P (Peptidyl): Growing polypeptide chain.

  • E (Exit): Discharged tRNA leaves.

Stages

1. Initiation: Small subunit binds mRNA; initiator tRNA pairs with $AUG$ start codon; large subunit joins.

2. Elongation:

   • Codon Recognition: tRNA-amino acid entry at A site.

   • Peptide Bond Formation: Catalyzed by localized $rRNA$.

   • Translocation: Ribosome shifts one codon toward $3'$ end.

3. Termination: Stop codon ($UAA, UAG, UGA$) recruitment of Release Factor; complex dissociates.

The Genetic Code

• Triplet: $64$ total codons ($4^3$).

• Degenerate: Multiple codons encode the same amino acid.

• Wobble: Flexibility in pairing at the third codon position.

• Universal: Nearly identical across all life forms.