Unit 5: RNA and Protein Synthesis and Processing - Subjective Questions

Transcription in prokaryotes is the synthesis of RNA from a DNA template, carried out by a single RNA polymerase enzyme.

Structure of RNA Polymerase (holoenzyme):

• Core enzyme:
• Sigma () factor: recognizes promoter sequences

Stages of Transcription:

1. Initiation:

   • The sigma factor guides the holoenzyme to the promoter region.
   • Prokaryotic promoters contain conserved consensus sequences: the -10 (Pribnow) box (TATAAT) and the -35 box (TTGACA).
   • The DNA unwinds to form an open complex.

2. Elongation:

   • After synthesizing a short RNA, the sigma factor is released.
   • The core enzyme moves along the template (), synthesizing RNA in the direction.

3. Termination:

   • Rho-dependent: Rho protein binds RNA and terminates transcription.
   • Rho-independent (intrinsic): A GC-rich hairpin loop followed by a poly-U stretch causes RNA release.

Key features: Transcription and translation are coupled in prokaryotes since there is no nuclear membrane.

Eukaryotic transcription occurs in the nucleus and is more complex than in prokaryotes, involving three distinct RNA polymerases.

Three RNA Polymerases:

• RNA Polymerase I: Synthesizes most rRNA (28S, 18S, 5.8S).
• RNA Polymerase II: Synthesizes mRNA and some snRNAs.
• RNA Polymerase III: Synthesizes tRNA, 5S rRNA, and small RNAs.

Assembly of Initiation Complex (for RNA Pol II):

1. TFIID binds the TATA box via its TBP (TATA-binding protein) subunit.
2. TFIIA and TFIIB stabilize the complex.
3. RNA Pol II with TFIIF joins.
4. TFIIE and TFIIH are recruited.
5. TFIIH has helicase activity (unwinds DNA) and kinase activity (phosphorylates the CTD, C-terminal domain).

Key features:

• Requires general transcription factors (GTFs).
• Requires the mediator complex and enhancers.
• The pre-mRNA undergoes extensive processing before export.
• Transcription and translation are spatially and temporally separated.

Comparison of Prokaryotic vs Eukaryotic Transcription:

Summary: Eukaryotic transcription is more regulated and compartmentalized, whereas prokaryotic transcription is streamlined for rapid gene expression.

Antibiotic inhibitors of transcription target RNA polymerase or DNA to block RNA synthesis.

Major Inhibitors:

• Rifampicin (Rifamycin):

  • Binds the -subunit of bacterial RNA polymerase.
  • Blocks initiation (prevents formation of the first phosphodiester bond).
  • Used to treat tuberculosis.

• Actinomycin D (Dactinomycin):

  • Intercalates into DNA at GC-rich regions.
  • Blocks elongation by preventing RNA polymerase movement.
  • Affects both prokaryotes and eukaryotes; used as an anticancer agent.

• -Amanitin:

  • Toxin from Amanita phalloides (death cap mushroom).
  • Inhibits eukaryotic RNA Polymerase II (and weakly Pol III).
  • Blocks translocation during elongation.

• Streptolydigin:

  • Binds bacterial RNA polymerase and inhibits elongation.

Clinical significance: Rifampicin's specificity for bacterial RNA polymerase makes it a selective antibacterial agent with minimal effect on human cells.

5' Capping is the first post-transcriptional modification of eukaryotic pre-mRNA, occurring co-transcriptionally when the transcript is about 25-30 nucleotides long.

Mechanism (formation of 7-methylguanosine cap):

1. RNA triphosphatase removes the terminal phosphate from the 5' end (leaving a diphosphate).
2. Guanylyltransferase adds a GMP via an unusual 5'-5' triphosphate linkage.
3. Guanine-7-methyltransferase adds a methyl group to position 7 of the guanine, forming the m7G cap (Cap 0).
4. Additional methylations on adjacent riboses give Cap 1 and Cap 2.

Structure: — a 7-methylguanosine linked by a triphosphate bridge.

Significance of the cap:

• Protects mRNA from degradation by 5' exonucleases.
• Essential for splicing of the first intron.
• Required for nuclear export.
• Recognized by eIF4E for translation initiation.
• Aids ribosome binding.

RNA splicing removes introns (non-coding sequences) and joins exons (coding sequences) in eukaryotic pre-mRNA.

Conserved Sequences:

• 5' splice site (donor): GU
• 3' splice site (acceptor): AG
• Branch point: An internal adenine (A) residue.
• Rule known as the GU-AG rule.

The Spliceosome:

• A large complex of snRNPs (small nuclear ribonucleoproteins): U1, U2, U4, U5, and U6.
• U1 binds the 5' splice site; U2 binds the branch point.

Two Transesterification Reactions:

1. First reaction: The 2'-OH of the branch-point adenine attacks the 5' splice site, forming a lariat structure.
2. Second reaction: The free 3'-OH of the upstream exon attacks the 3' splice site, joining the two exons and releasing the intron lariat.

Significance:

• Enables alternative splicing, producing multiple proteins from one gene.
• Increases protein diversity.

Note: Some RNAs (Group I and II introns) are self-splicing ribozymes.

Polyadenylation is the addition of a poly(A) tail (approximately 200 adenine residues) to the 3' end of eukaryotic pre-mRNA.

Mechanism:

1. Recognition of the polyadenylation signal (AAUAAA) located ~10-30 nucleotides upstream of the cleavage site.
2. CPSF (Cleavage and Polyadenylation Specificity Factor) binds the AAUAAA signal.
3. CstF (Cleavage Stimulation Factor) binds the downstream GU-rich element.
4. The pre-mRNA is cleaved at the poly(A) site.
5. Poly(A) polymerase (PAP) adds adenine residues without a template.
6. PABP (Poly(A)-Binding Protein) coats the tail.

Biological Importance:

• Stabilizes mRNA against exonuclease degradation.
• Facilitates nuclear export.
• Enhances translation efficiency (circularization of mRNA via PABP-eIF4G).
• Determines mRNA half-life.

Note: Histone mRNAs are a notable exception — they generally lack a poly(A) tail.

Prokaryotic translation occurs on 70S ribosomes (30S + 50S subunits).

1. Initiation:

• The Shine-Dalgarno sequence in mRNA aligns with the 16S rRNA of the 30S subunit.
• Initiator tRNA carries N-formylmethionine (fMet).
• Initiation factors: IF1, IF2, IF3.
• fMet-tRNA binds the start codon AUG at the P site.

2. Elongation:

• EF-Tu delivers aminoacyl-tRNA to the A site (GTP-dependent).
• Peptidyl transferase (a ribozyme in 23S rRNA) forms the peptide bond.
• EF-G catalyzes translocation (GTP-dependent), moving the ribosome one codon.

3. Termination:

• Stop codons (UAA, UAG, UGA) are recognized by release factors RF1, RF2, RF3.
• The polypeptide is released and the ribosome dissociates (with help from RRF).

Key feature: Translation is coupled with transcription.

Eukaryotic translation occurs on 80S ribosomes (40S + 60S subunits).

1. Initiation:

• Initiator tRNA carries methionine (Met), not formylmethionine.
• The 43S pre-initiation complex (40S + eIF2-GTP-Met-tRNA) forms.
• eIF4E binds the 5' cap; eIF4G and eIF4A aid scanning.
• Ribosome scans from the 5' cap to the first AUG (Kozak sequence).
• Many eukaryotic initiation factors (eIFs) are involved.

2. Elongation:

• eEF1 delivers aminoacyl-tRNA; eEF2 catalyzes translocation.
• Peptidyl transferase (28S rRNA) forms peptide bonds.

3. Termination:

• Single release factor eRF1 recognizes all stop codons; eRF3 provides GTP hydrolysis.

Differences from Prokaryotes:

Translation inhibitors target ribosomes and are widely used as antibiotics and research tools.

Inhibitors of Prokaryotic Translation (Antibacterial):

• Streptomycin: Binds 30S subunit; causes misreading of mRNA and inhibits initiation.
• Tetracycline: Blocks the A site on the 30S subunit, preventing aminoacyl-tRNA binding.
• Chloramphenicol: Inhibits peptidyl transferase on the 50S subunit.
• Erythromycin: Binds 50S subunit; blocks translocation (exit tunnel).
• Aminoglycosides (e.g., kanamycin): Cause misreading.

Inhibitors of Eukaryotic Translation:

• Cycloheximide: Inhibits peptidyl transferase on the 60S subunit; blocks elongation.
• Diphtheria toxin: ADP-ribosylates and inactivates eEF2, blocking translocation.

Inhibitors of Both:

• Puromycin: Structural analog of aminoacyl-tRNA; causes premature chain termination by incorporating into the growing peptide.

Clinical relevance: The structural differences between 70S and 80S ribosomes allow selective targeting of bacteria without harming host cells.

Post-translational modifications (PTMs) are chemical or structural changes made to a protein after its synthesis on the ribosome. They expand the functional diversity of the proteome.

Major Categories:

1. Chemical modifications: Addition of chemical groups (phosphorylation, glycosylation, etc.).
2. Proteolytic cleavage: Removal of specific peptide segments.
3. Protein splicing: Removal of internal inteins.

Importance of PTMs:

• Regulate enzyme activity (e.g., phosphorylation switches enzymes on/off).
• Control protein localization (e.g., lipidation targets proteins to membranes).
• Influence protein stability and turnover (e.g., ubiquitination for degradation).
• Mediate protein-protein interactions and signaling.
• Enable proper folding and maturation (e.g., disulfide bond formation).
• Increase structural and functional diversity beyond the genetic code.

Significance: A single gene can give rise to many functionally distinct protein forms (proteoforms) through PTMs.

Chemical PTMs involve the covalent addition of chemical groups to specific amino acid residues.

Major Types:

• Phosphorylation:

  • Addition of a phosphate group to Ser, Thr, or Tyr by kinases.
  • Reversible (removed by phosphatases); central to signal transduction.

• Glycosylation:

  • N-linked (to asparagine) or O-linked (to Ser/Thr).
  • Important for protein folding, stability, and cell recognition.

• Methylation:

  • Addition of methyl groups to Lys or Arg; important in histone regulation.

• Acetylation:

  • Addition of acetyl groups to Lys; regulates chromatin structure and gene expression.

• Ubiquitination:

  • Attachment of ubiquitin to Lys; tags proteins for proteasomal degradation.

• Lipidation:

  • Addition of lipid groups (prenylation, myristoylation, palmitoylation); anchors proteins to membranes.

• Hydroxylation:

  • Addition of -OH (e.g., to proline in collagen).

• Disulfide bond formation:

  • Covalent linkage between cysteine residues; stabilizes structure.

Proteolytic cleavage is the irreversible removal of specific peptide segments from a protein by proteases, often converting an inactive precursor into an active form.

Types of Precursors:

• Pre-proteins: Contain signal sequences.
• Pro-proteins: Contain propeptides that must be removed.
• Pre-pro-proteins: Contain both.

Examples:

1. Insulin:

   • Synthesized as preproinsulin proinsulin (after signal removal) insulin (after removal of C-peptide).
   • Mature insulin has A and B chains linked by disulfide bonds.

2. Zymogens (proenzymes):

   • Trypsinogen trypsin; chymotrypsinogen chymotrypsin.
   • Prevents premature enzyme activity that could damage the cell.

3. Blood clotting factors:

   • Prothrombin thrombin during coagulation cascade.

4. Signal peptide removal:

   • Signal peptidase cleaves N-terminal signal sequences for secretion.

Significance: Provides rapid, irreversible activation and controls timing and location of protein activity.

Protein splicing is an autocatalytic post-translational process in which an internal protein segment (intein) is excised, and the flanking segments (exteins) are joined by a peptide bond.

Key Components:

• Intein: The internal segment that is removed (analogous to introns in RNA).
• Exteins: The N-extein and C-extein flanking regions that are joined to form the mature protein (analogous to exons).

Mechanism (four steps):

1. N-S (or N-O) acyl shift: A rearrangement at the N-terminal splice junction forms a (thio)ester.
2. Transesterification: The side chain nucleophile of the first C-extein residue attacks, transferring the N-extein to the C-terminus.
3. Asparagine cyclization: Cleaves the intein-C-extein bond, releasing the intein.
4. S-N (or O-N) acyl shift: Forms a stable peptide bond between the two exteins.

Features:

• It is self-catalytic (no external enzyme required).
• Inteins often contain a homing endonuclease domain.
• Found in bacteria, archaea, and some lower eukaryotes.

Applications: Used in protein engineering and expressed protein ligation.

Comparison of RNA Splicing vs Protein Splicing:

Common theme: Both processes remove internal segments and join flanking regions, but they operate at different molecular levels (RNA vs protein).

Alternative splicing is a process by which a single pre-mRNA can be spliced in different ways to produce multiple mRNA variants and hence different protein isoforms.

Types of Alternative Splicing:

• Exon skipping (cassette exon): An exon is included or excluded.
• Intron retention: An intron is retained in the mature mRNA.
• Alternative 5' splice site selection.
• Alternative 3' splice site selection.
• Mutually exclusive exons: Only one of two exons is retained.

Regulation:

• Controlled by SR proteins (splicing enhancers) and hnRNP proteins (splicing silencers).
• Depends on cis-acting elements (ESE, ESS, ISE, ISS).

Significance:

• Enables one gene to encode many proteins (e.g., the Dscam gene can produce thousands of isoforms).
• Explains how ~20,000 human genes produce a much larger proteome.
• Provides tissue-specific and developmental regulation.
• Errors in splicing are linked to diseases (e.g., -thalassemia, cancer).

A mature eukaryotic mRNA is a highly processed molecule optimized for stability and efficient translation.

Structural Features (5' to 3'):

1. 5' Cap (m7G cap):

   • 7-methylguanosine linked via 5'-5' triphosphate bridge.
   • Protects against degradation and aids ribosome binding.

2. 5' Untranslated Region (5' UTR):

   • Non-coding; contains the Kozak sequence near the start codon.

3. Coding Region (ORF):

   • Begins with AUG (start codon) and ends with a stop codon (UAA/UAG/UGA).

4. 3' Untranslated Region (3' UTR):

   • Contains regulatory elements and the AAUAAA polyadenylation signal.

5. Poly(A) Tail:

   • ~200 adenine residues; enhances stability, export, and translation.

Processing Steps:

• 5' Capping (co-transcriptional)
• Splicing (intron removal)
• 3' Cleavage and Polyadenylation
• RNA editing (in some cases)

Function: Serves as the template that is exported to the cytoplasm and translated into protein, with each feature contributing to stability and translational efficiency.

Eukaryotic translation initiation is a highly regulated, cap-dependent process involving many eukaryotic initiation factors (eIFs).

Steps:

1. Ternary complex formation:

   • eIF2-GTP binds Met-tRNAi to form the ternary complex.

2. 43S pre-initiation complex:

   • The ternary complex joins the 40S subunit with eIF1, eIF1A, eIF3, and eIF5.

3. Cap recognition:

   • eIF4F complex (eIF4E + eIF4G + eIF4A) binds the 5' cap.
   • eIF4E recognizes the m7G cap; eIF4A is an RNA helicase; eIF4G is a scaffold.

4. Scanning:

   • The 43S complex is recruited to the 5' end and scans along the mRNA (ATP-dependent) until it finds the first AUG in a Kozak context (gccRccATGG).

5. 48S complex and joining:

   • Codon-anticodon pairing triggers eIF2-GTP hydrolysis (via eIF5).
   • Initiation factors are released.
   • eIF5B-GTP promotes joining of the 60S subunit to form the 80S initiation complex.

Regulation: Phosphorylation of eIF2 and availability of eIF4E are major control points.

Termination of prokaryotic transcription occurs by two mechanisms:

1. Rho-Independent (Intrinsic) Termination:

• Relies solely on specific RNA sequences.
• Features:
  • A GC-rich inverted repeat forms a stable hairpin (stem-loop) structure.
  • Followed by a stretch of poly-U (uracil) residues.
• The hairpin causes RNA polymerase to pause, and the weak rU-dA base pairs allow the transcript to dissociate.
• No protein factor is required.

2. Rho-Dependent Termination:

• Requires the Rho protein (an ATP-dependent helicase).
• Features:
  • Rho binds a specific C-rich sequence called the rut site (rho utilization site) on the RNA.
  • Rho moves along the RNA toward the polymerase.
  • When the polymerase pauses, Rho catches up and uses its helicase activity to unwind the RNA-DNA hybrid, releasing the transcript.

Comparison Table:

Aminoacylation (tRNA charging) is the process of attaching the correct amino acid to its corresponding tRNA, catalyzed by aminoacyl-tRNA synthetases.

Reaction (Two Steps):

1. Amino acid activation:
   

   • Forms a high-energy aminoacyl-adenylate intermediate.

2. Transfer to tRNA:
   

   • The amino acid is attached to the 3' end (CCA) of the tRNA.

Aminoacyl-tRNA Synthetases:

• Each amino acid has a specific synthetase.
• Class I: attach amino acid to 2'-OH; usually monomeric.
• Class II: attach to 3'-OH; usually dimeric.

Importance of Fidelity:

• The ribosome cannot verify whether the correct amino acid is attached — it only reads the codon-anticodon pairing.
• An incorrectly charged tRNA leads to misincorporation of amino acids.
• Synthetases have proofreading (editing) sites to hydrolyze incorrectly charged products.
• This ensures the second genetic code (amino acid–tRNA matching) is accurate, maintaining protein integrity.