RNA vs DNA: Types and Post-Transcriptional Modifications

RNA vs. DNA

• RNA is synthesized in the same manner as DNA, in the 5' to 3' direction.
• It is made using DNA as a template.
• RNA has three major differences from DNA:
  • RNA contains ribose sugar, while DNA has deoxyribose.
  • RNA uses uracil instead of thymine (along with A, C, and G).
  • RNA is typically single-stranded, while DNA is double-stranded.

Types of RNA Molecules

• All RNA molecules are structurally similar but serve different functions.
• mRNA serves as a template for protein synthesis.
  • The sequence of nucleotides in mRNA determines the amino acid sequence in the protein.
• tRNA is attached to specific amino acids.
  • It guides amino acids to the ribosome and directs their addition into the growing polypeptide.
• rRNA, along with many other proteins, is present in the ribosome.
  • It is necessary for proper ribosome assembly and function.

Post-Transcriptional Modifications (Eukaryotes)

• Each mRNA molecule is modified after transcription to be recognized by the ribosome and protected from degradation.
• Two structures are added:
  • A 5' cap structure.
  • A 3' poly(A) tail.
• In Eukaryotes, mRNA must be spliced, meaning introns are removed, and exons stay in.
• A protein complex called the spliceosome recognizes the junction between introns and exons, binds to two junctions, and removes the intron as a loop.

Translation (Protein Synthesis)

• Requires a ribosome, mRNA, and tRNA molecules linked to amino acids (amino acyl-tRNA).
• mRNA is read by a ribosome.

Amino Acyl-tRNA

• It is the adapter that translates a triplet codon in the mRNA to an amino acid in the protein.

Codons

• mRNA is read in groups of three nucleotides, called a codon.
• There are three ways to read nucleotides in groups of three, called reading frames.

Ribosome Start Site

• Prokaryotes: the ribosome binds to a specific RNA sequence known as the Shine-Dalgarno sequence.
  • Each gene has its own Shine-Dalgarno sequence.
• Eukaryotes: the ribosome binds to the 5' end of the mRNA.
  • Each gene is on its own mRNA.
• Both: the ribosome slips along the mRNA sequence toward the 3' end until it encounters a start codon (AUG).
  • The first AUG defines the reading frame.

tRNA

• It is an "adapter" molecule that must be used to associate an amino acid with its corresponding codon.

Anticodon

• The anticodon of tRNA binds to the mRNA codon by complementary base pairing.
• Wobble position is where unusual base pairing is permitted: G:U and I:A, I:C, I:U.

Translation Initiation

• Initiation factors and GTP bind to the 30S ribosomal subunit.
• Initiator tRNA and mRNA then bind to the 30S ribosomal subunit.
• The 50S ribosomal subunit becomes bound to the 30S initiation complex.

Translation Elongation

• It involves elongation factors, especially EF-Tu, which delivers the charged tRNA to the A-site, and EF-G, which moves the tRNAs during translocation.
• It costs 3 GTP per amino acid.
  • One hydrolyzed by EF-Tu provides energy for the peptide bond.
  • One hydrolyzed by EF-G is used for translocation.
• Has two reactions:
  • Transpeptidation (or peptidyl transferase).
  • Translocation (or mRNA movement).
• The Elongation reaction takes place in the peptidyl transferase center of the ribosome.

Translation Termination

• No tRNAs match the STOP codons, causing the ribosome to stall.
• A "Release Factor" binds instead of tRNA and causes the ribosome to fall apart.
• Sometimes a tRNA recognizes a stop codon, referred to as “read-through”.
  • The sequence beyond the STOP codon is random and will contain other stop codons (avg. 3/64 → stop codon is approximately every 21 codons).
• Prokaryotes can make many proteins in an "assembly line" fashion from the same mRNA before the RNA is even finished.
• Coupled transcription and translation occur in bacteria.

Antibiotics and Protein Synthesis

• Many antibiotics target protein synthesis:
  • Streptomycin: blocks initiation.
  • Tetracycline: blocks elongation.
  • Chloramphenicol: prevents transpeptidation.
  • Clindamycin: prevents transpeptidation.
  • Erythromycin: blocks translocation.
  • Mupirosin: inhibits isoleucyl-tRNA synthetase.

DNA Change

• Even small changes in the coding region of a gene can affect H-bonding in the protein and alter protein function.
  • Point mutations alter one codon, leading to a change in an amino acid.
  • Insert/delete mutations change the reading frame.
• Promoter alteration can lead to increased or decreased transcription.
  • Changes the number of copies of protein in the cell.
• If the protein is an enzyme, its function can be lost, resulting in a mutant metabolic phenotype.