Transcription and translation

Transcription

tRNA and rRNA: composition and roles in translation:

• Although transfer RNA (tRNA) and ribosomal RNA (rRNA) result from transcription, they do not function as the direct template for translation (unlike mRNA). Instead, tRNA brings the correct amino acid to the ribosome during protein synthesis, while rRNA is a central part of the ribosomal structure responsible for catalyzing the formation of peptide bonds.

tRNA structure and function:

• Nucleotide composition: tRNA contains numerous chemically modified nucleotides that enhance stability and functionality.
• Cloverleaf configuration: tRNA typically forms a cloverleaf structure with an anticodon loop (which base-pairs with the mRNA codon) and an amino acid attachment site at the 3′ end. The amino acid connects to the tRNA’s 3′-OH group via an ester linkage.

rRNA structure and function:

• Nucleotide composition: rRNA also contains modified nucleotides that ensure proper folding and catalytic activity.
• Catalytic role: The large ribosomal subunit’s rRNA houses the active site that catalyzes peptide bond formation. Remarkably, rRNA alone can perform this reaction even without associated ribosomal proteins, establishing rRNA as a ribozyme.

Transcription entails synthesizing RNA from a DNA template and requires:

• RNA polymerase and promoters: The enzyme RNA polymerase binds to a promoter region (e.g., a TATA box) in double-stranded DNA. Initially, the DNA remains closed, but then it unwinds to form an open complex.
• Chain initiation: No primer is required (unlike DNA replication). Once RNA polymerase docks onto the promoter, it can begin adding ribonucleotides complementary to the DNA template strand.
• Chain elongation: Nucleoside triphosphates (A, U, G, and C) are incorporated in the 5′ $\to$ 3′ direction, extending the newly forming RNA molecule.
• Chain termination:
  • Intrinsic termination: A specific sequence leads to a stem-loop structure in the RNA, causing it to detach from the template.
  • Rho-dependent termination: The ρ factor (rho protein) pursues the RNA polymerase along the nascent RNA and dislodges it from the DNA strand.

mRNA processing in eukaryotes:
In eukaryotic cells, mRNA undergoes post-transcriptional modifications to stabilize it and prepare it for export to the cytoplasm:

• 5′ cap: A modified guanine nucleotide attached in an atypical manner at the mRNA’s 5′ end, preventing exonuclease degradation and promoting ribosomal binding.
• 3′ poly-A tail: Several adenine residues are appended to the 3′ end, preventing rapid breakdown and aiding in nuclear export.
• Splicing: Non-coding regions (introns) are removed, and coding segments (exons) are spliced together. Alternative splicing allows for various mRNA isoforms from the same gene.

Prokaryotic mRNA generally lacks these 5′ cap and poly-A tail features, and in some bacteria, transcription and translation occur simultaneously.

Ribozymes, spliceosomes, and RNA-associated complexes:

• Ribozymes: RNA enzymes capable of catalysis. The best-known example is the rRNA in the large ribosomal subunit.
• Spliceosomes: Large complexes of RNA and protein responsible for mRNA splicing. They can contain ribozyme-like activity through their RNA components.
• snRNPs (small nuclear ribonucleoproteins): Subunits that combine with RNA and proteins to form spliceosomes.
• snRNAs (small nuclear RNAs): The RNA portion of snRNPs, essential for recognizing splicing sites and catalyzing intron removal.

Functional and evolutionary importance of introns

• Functional roles: Introns can facilitate alternative splicing, allowing a single gene to produce multiple protein variants and also influencing gene regulation.
• Evolutionary perspective: Introns may have originated from mobile genetic elements and have persisted because they allow for greater genomic flexibility and complex gene regulation.

Translation

Roles of mRNA, tRNA, and rRNA

• Messenger RNA (mRNA) carries the genetic code from DNA, with each three-nucleotide codon specifying a particular amino acid in the resulting polypeptide.
• Transfer RNA (tRNA) bears an anticodon that pairs with the corresponding codon on mRNA, and an amino acid attached at the 3′ end by an ester linkage. This ensures that the correct amino acid is inserted whenever its codon appears on the mRNA.
• Ribosomal RNA (rRNA) constitutes the core of the ribosome, the complex that catalyzes peptide bond formation. The large subunit’s rRNA plays a catalytic (ribozyme-like) role in connecting amino acids, while the small subunit’s rRNA helps recognize and position the mRNA.

Ribosome structure and function:
A ribosome acts as the site where translation—the synthesis of proteins—occurs. It consists of two distinct subunits (large and small) that unite around the mRNA:

1. Large subunit: Responsible for forming peptide bonds (peptidyl transfer).
2. Small subunit: Engages with the mRNA, identifying sequences such as the Shine-Dalgarno region in prokaryotes or the Kozak sequence in eukaryotes.

When aligned, the two subunits create a “sandwich” structure, holding the mRNA and incoming tRNAs in place to enable amino acid addition.

Mechanism of translation: initiation, elongation, and termination:

1. Chain initiation:
   • In prokaryotes, the small ribosomal subunit attaches near the Shine-Dalgarno sequence on the mRNA (in eukaryotes, it targets the Kozak sequence).
   • An initiator tRNA (often carrying formyl-methionine, fMet, in prokaryotes) binds to the start codon (AUG).
   • Initiation factors and GTP facilitate the assembly of the complete ribosome, placing the tRNA at the P site to begin translation.
2. Chain elongation:
   • Aminoacyl-tRNA binding: A new tRNA carrying its amino acid (aminoacyl-tRNA) arrives at the A site, aided by elongation factors and GTP.
   • Peptidyl transfer: The ribosome’s large subunit catalyzes the bond between the existing chain in the P site and the amino acid in the A site, effectively transferring the growing peptide to the A-site tRNA.
   • Translocation: The ribosome advances one codon along the mRNA, shifting the peptidyl-tRNA from the A site to the P site and moving the now-empty tRNA to the E site, where it exits. The A site is then available for a new aminoacyl-tRNA.
3. Chain termination:
   • A stop codon (UAA, UAG, or UGA) appearing in the A site prompts the entry of release factors (bound to GTP) instead of a tRNA.
   • The completed polypeptide is severed from the final tRNA in the P site. Subsequently, the ribosome complex dissociates, freeing the newly synthesized protein.

Amino acid activation:

• Before translation, specialized enzymes called aminoacyl-tRNA synthetases couple the correct amino acid to its corresponding tRNA. This process requires ATP and ensures each tRNA is charged with the right amino acid.

Post-translational modification of proteins
After translation, proteins often undergo additional modifications, including:

• Glycosylation: Attaching carbohydrate groups for targeting or structural functions (e.g., in proteoglycans).
• Lipid addition: Adding fatty groups for membrane anchoring.
• Acetylation: Tagging that can direct subcellular localization.
• Disulfide bond formation: Linking separate protein subunits, stabilizing tertiary or quaternary structures.
• Phosphorylation: Activating or deactivating enzymes.
• Ubiquitination: Marking proteins for degradation.