Molecular Biology: Transcription, RNA Processing, and the Mechanism of Translation

Comparison of Transcription and RNA Processing

Prokaryotes:
- The process involves DNA being transcribed directly into mRNA ( $messenger\,RNA$ ).
- Translation of mRNA into a protein occurs shortly after or even during transcription.
Eukaryotes:
- Transcription occurs in the nucleus to produce a transcription unit known as the "primary RNA transcript" or pre-mRNA.
- The primary transcript contains both exons (coding sequences) and introns (non-coding intervening sequences).
- RNA Processing Steps:
  1. $5'$ Capping: A cap is added to the $5'$ end of the RNA.
  2. RNA Splicing: Introns are removed and exons are joined together.
  3. $3'$ Polyadenylation: A poly-A tail is added to the $3'$ end.
- The finished mRNA is exported from the nucleus to the cytoplasm.
- Translation occurs in the cytoplasm to produce a protein.

The Principles of the Genetic Code

Codons:
- Genetic information is encoded as a sequence of non-overlapping base triplets.
- Each codon (word) consists of 3 bases (letters) that code for one specific amino acid.
Mathematical Foundations:
- With 4 different bases ( $A, U, C, G$ ) and 3 positions in each codon, there are $4 \times 4 \times 4 = 64$ possible codons.
Coding Characteristics:
- Redundancy (Degeneracy): There are $64$ codons but only $20$ amino acids; therefore, multiple "synonymous" codons can code for the same amino acid.
- Start Signal: One codon ( $AUG$ ) serves as the START signal and also codes for Methionine. It is found at the beginning of every protein.
- Stop Signals: Three codons ( $UAA, UAG, UGA$ ) serve as STOP signals to mark the end of the protein sequence.
- Unambiguity: The code is unambiguous; one specific codon never codes for more than one amino acid.
- Universality: The code is universal, used by almost all living organisms.
DNA-RNA Correspondence:
- The genetic code refers to the bases in mRNA.
- The coding DNA strand has the identical sequence to the mRNA (with $T$ instead of $U$ ).
- The template DNA strand, from which mRNA is transcribed, has a complementary sequence (with $T$ instead of $U$ ).
- Example: Template DNA $3'-AAA-5'$ corresponds to mRNA $5'-UUU-3'$ and coding DNA $5'-TTT-3'$ , all of which specify the amino acid Phenylalanine.

The Adapter Molecule: Transfer RNA (tRNA)

The Adapter Hypothesis:
- Proposed by Francis Crick, suggesting a "bridge" molecule that binds amino acids and recognizes nucleotide sequences.
Structure of tRNA:
- Composed of a single RNA strand approximately $80$ nucleotides long.
- Anticodon: A specific triplet that pairs with the mRNA codon via hydrogen bonding.
- Amino Acid Attachment Site: Located at the $3'$ end, ending in the sequence $CCA$ with a hydroxyl ( $OH$ ) group.
- Two-dimensional structure resembles a "cloverleaf," while the three-dimensional structure is more complex.
Charging of tRNA (Aminoacylation):
- Performed by a group of highly specific enzymes called Aminoacyl-tRNA-synthetases.
- Uses $ATP$ to link the tRNA to its specific amino acid via a high-energy bond.
- Chemical Reactions:
  1. $amino\,acid + ATP \rightarrow aminoacyl-AMP + PPi$
  2. $aminoacyl-AMP + tRNA \rightarrow aminoacyl-tRNA + AMP$
Wobble Base Pairing:
- Strict specificity for the base at the $3'$ end of the codon is not always required.
- Allows cells to function with fewer types of tRNA species.
- Involves modified purine bases like Inosine ( $I$ ).
- The code remains unambiguous despite wobble pairing.

Ribosome Structure and Catalyst Function

Function:
- Ribosomes facilitate the specific pairing of tRNA anticodons with mRNA codons and catalyze peptide bond formation.
- They are non-specific; any ribosome can translate any mRNA to create any protein.
Composition:
- Consist of a large subunit and a small subunit.
- Composed of ribosomal RNA ( $rRNA$ ) and dozens of proteins arranged in a precise pattern.
- Subunits are held together by ionic and hydrophobic forces, not covalent bonds.
- Subunits remain separate when not actively translating.
Sedimentation and Size:
- Mass is several million Daltons.
- Measured in Svedberg units ( $S$ ), a non-SI metric unit for sedimentation coefficients based on how fast a particle settles under acceleration.
Ribosomal Binding Sites (Large Subunit):
- A Site (Aminoacyl): Where the charged tRNA anticodon binds to the mRNA codon.
- P Site (Peptidyl): Where the tRNA adds its amino acid to the growing polypeptide chain.
- E Site (Exit): Where the uncharged tRNA resides before release.
Catalytic Activity:
- Peptidyl transferase activity is performed by the $rRNA$ of the large subunit, not a protein.
- This provides evidence for the "RNA World" hypothesis, suggesting catalytic RNA evolved before DNA.
Prokaryotic vs. Eukaryotic Ribosomes:
- Prokaryotes: $3$ rRNAs + $49$ proteins (Large) and $1$ rRNA + $33$ proteins (Small).
- Differences in proteins and RNAs allow antibiotics to target prokaryotic ribosomes without harming eukaryotic hosts.
- 2009 Nobel Prize in Chemistry: Awarded to Venkatraman Ramakrishnan, Thomas A. Steitz, and Ada E. Yonath for studies on ribosome structure and function.

The Mechanism of Translation

Initiation:
- Requires an initiation complex: small subunit + charged tRNA + mRNA.
- Prokaryotes: Small subunit binds the Shine-Dalgarno sequence ( $AGGAGG$ ), located $10\,bp$ upstream of the start codon.
- Eukaryotes: Small subunit binds the $5'$ cap and scans the mRNA until it finds the $AUG$ start codon.
- Methionine-charged tRNA binds the start codon, and the large subunit joins so the tRNA occupies the P site. This process requires proteins called initiation factors.
Elongation:
- A charged tRNA enters the A site. The small subunit $rRNA$ validates the hydrogen bonding match.
- Peptidyl Transferase Activity:
  1. Breaks the bond between the tRNA in the P site and its amino acid.
  2. Forms a peptide bond between that amino acid and the one on the tRNA in the A site.
- Translocation: The ribosome shifts one codon in the $5'$ to $3'$ direction. The tRNA in the A site moves to the P site ( $A \rightarrow P$ ), and the uncharged tRNA moves to the E site ( $P \rightarrow E$ ) to be released.
- The cycle is assisted by elongation factors.
Termination:
- Occurs when a stop codon ( $UAA, UAG, UGA$ ) enters the A site.
- A protein release factor (not a tRNA) binds and hydrolyzes the bond between the polypeptide and the tRNA in the P site.
- The polypeptide is released ( $N-terminus$ is the first amino acid, initially Methionine; $C-terminus$ is the last).

Polysomes and Post-Translational Events

Polysomes (Polyribosomes):
- Several ribosomes can simultaneously translate a single mRNA, creating a "beads on a string" appearance and producing multiple polypeptide copies.
Polypeptide Destinations and Signaling:
- Folding is determined by the amino acid sequence; chaperonines may assist.
- Signal Sequences (Signal Peptides): Direct proteins to destinations (e.g., ER, nucleus, organelles).
- Nuclear Localization Signal (NLS): Short sequence like $-Pro-Pro-Lys-Lys-Lys-Arg-Lys-Val-$ targets proteins to the nucleus post-translation.
- ER Targeting: Translation pauses, the signal sequence binds a receptor on the ER surface, and the polypeptide moves through a channel into the organelle.
Post-Translational Modifications:
- Proteolysis: Cutting the polypeptide chain with proteases (e.g., removing signal sequences).
- Glycosylation: Adding sugars in the ER/Golgi to form glycoproteins (important for targeting, stability, and recognition).
- Phosphorylation: Adding phosphate groups via kinases to change protein conformation and activity (e.g., signaling or enzyme activation).

Summary of Start and Stop Signals

Transcription:
- Initiation: Promoter sequence in DNA.
- Termination: Terminator sequence in DNA.
Translation:
- Initiation: $AUG$ start codon in mRNA.
- Termination: $UAA, UAG, \text{or } UGA$ stop codon in mRNA.