Comprehensive Study Notes on Protein Translation

Overview of Translation

• Translation is defined as the final step in gene expression, involving the production of specific proteins by decoding the mRNA created during transcription.

• The fundamental requirements for translation include:

  • All three major forms of RNA: mRNA, tRNA, and rRNA.

  • The 20 basic amino acids.

  • Energy, which is required because the process involves the formation of a polymer.

  • Specific proteins to perform the work of polymerization.

• Within the process, proteins are found in ribosomes, where they are held in the correct orientation by RNA.

The Genetic Code and mRNA Decoding

• The genetic code consists of the sequence of bases found on the sense strand regions of DNA.

• This coding information is reproduced in the form of mRNA, which allows the code to exit the nucleus and enter the cytoplasm.

• In the cytoplasm, the code is decoded to dictate the specific sequence of amino acids used to construct a polypeptide.

• The mRNA is decoded by reading three bases at a time; this sequence of three bases is referred to as a codon.

• The ribosome reads the mRNA in the $5' \rightarrow 3'$ direction and writes the protein in the direction of $NH_3 \rightarrow COOH$ ($N \rightarrow C$ terminus).

Mathematical Basis of the Genetic Code

• Each codon consists of 3 slots, and each slot can be filled by 1 of 4 possible bases ($A$, $G$, $U$, or $C$).

• The number of possible unique codons is calculated as:   $4 \times 4 \times 4 = 64$

• Distribution of the 64 codons:

  • 61 codons code for specific amino acids.

  • 3 codons are stop codons, which signal the termination of translation.

Transfer RNA (tRNA) Structure and Function

• There are at least 20, and up to 64, different tRNA molecules to account for the various possible codons.

• tRNA is single-stranded, but it exhibits significant internal base-pairing, with approximately $50\%$ of the structure existing as a double-helix.

• Key features of the tRNA molecule:

  • The $3'$ end serves as the site for the attachment of one specific amino acid.

  • The anticodon region can base pair with the mRNA codon and serves as the "decoder."

  • The anticodon portion determines which amino acid is attached to the $3'$ end.

• tRNA contains unusual bases beyond $A$, $G$, $U$, and $C$. For example, Inosine comprises approximately $20\%$ of all bases in tRNA.

Aminoacyl-tRNA Synthetases and Activation

• The DNA code is described as "degenerate" because a single codon codes for only one amino acid, but one amino acid can be associated with several different codons.

• The attachment of the correct amino acid to the tRNA with the matching anticodon is performed by a family of enzymes called aminoacyl-tRNA synthetases.

• Each enzyme type is specific for one tRNA and its matching amino acid.

• The activation of tRNA is a two-step reaction catalyzed by aminoacyl-tRNA synthetase and energized by ATP:

  1. $AA + ATP \rightarrow \text{AA-AMP} + PP_i$

  2. $\text{AA-AMP} + tRNA \rightarrow \text{AA-tRNA} + AMP$

• The resulting molecule, consisting of a tRNA with an amino acid bound to it, is called an aminoacyl-tRNA.

Ribosome Composition and Architecture

• Ribosomes are composed of two subunits: one large and one small. These exist independently in the cytoplasm until translation is initiated, at which point they assemble on the mRNA.

• The structure of ribosomes differs between eukaryotes and prokaryotes:

  • Eukaryotic Ribosome (80S):

  • Large Subunit (60S): Contains 28S and 5.8S rRNA, and approximately 50 proteins.

  • Small Subunit (40S): Contains 18S rRNA and approximately 30 proteins.

  • Prokaryotic Ribosome (70S):

  • Large Subunit (50S): Contains 23S and 5S rRNA, and approximately 30 proteins.

  • Small Subunit (30S): Contains 16S rRNA and approximately 30 proteins.

• Role of rRNA: The RNAs hold the ribosomal proteins in the precise positions required for translation to occur.

• Physiological Context: A single bacterial cell has approximately $20,000$ ribosomes, making up about $25\%$ of the cell mass, illustrating that protein synthesis is a highly active and critical process.

Initiation of Translation

• Translation begins at the start codon, which is always $A-U-G$ on mRNA.

• In prokaryotes (specifically E. coli), there is a Shine-Dalgarno (SD) sequence located about 6-10 bases on the $5'$ side of the start codon. The sequence is $5'\text{-AGGAGG-}3'$.

• The SD sequence base pairs with a portion of the 16S rRNA within the small (30S) ribosomal subunit.

• Initiation Factors (IFs):

  • In prokaryotes, there are 3 factors: IF1, IF2, and IF3, which are bound to the 30S subunit to promote attachment to mRNA.

  • IF2 (or eIF2 in eukaryotes) is vital for the recognition of the AUG codon by the initiator tRNA.

  • In eukaryotes, there are at least 9 initiation factors (abbreviated as eIF).

• Initiator tRNA:

  • In eukaryotes, the initiator tRNA binds Methionine (Met).

  • In prokaryotes, Methionine is formylated by the enzyme transformylase (using $N^{10}\text{-formyltetrahydrofolate}$ as a carbon donor) to yield formylmethionine (fMet).

  • The reaction is: $\text{Met-tRNA} + \text{Formyl-THF} \rightarrow \text{fMet-tRNA} + \text{THF}$.

• Antibiotic interference: Streptomycin binds to the 30S subunit and blocks initiation in prokaryotes.

• Completion of initiation occurs through:

  1. Binding of met-tRNA (or fMet-tRNA).

  2. Release of IF1, IF2, and IF3.

  3. Hydrolysis of GTP ($GTP \rightarrow GDP + P_i$).

  4. Attachment of the large (50S) subunit.

Ribosomal Domains and the A, P, and E Sites

• The assembled ribosome features three distinct domains:

  • P Site (Peptidyl): The region where the initiator tRNA currently projects or where the growing polypeptide chain is held.

  • A Site (Aminoacyl): Immediately adjacent to the P site, where the next codon resides and the incoming aminoacyl-tRNA enters.

  • E Site (Exit): Located to the left of the P site, where discharged tRNAs exit the ribosome.

Elongation of the Polypeptide Chain

• Elongation involves three repeating steps:

  1. Binding of the next Aminoacyl-tRNA: The next tRNA is brought to the A site. This requires elongation factor EF-Tu (in prokaryotes) or eEF-1 (in eukaryotes) and the hydrolysis of GTP.

  2. Peptide Bond Formation: The enzyme Peptidyl Transferase (a component of the large ribosomal subunit) catalyzes the formation of a peptide bond between the amino acid at the A site and the one at the P site.

  3. Translocation: The ribosome moves 3 bases down the mRNA. This requires elongation factor EF-G (in prokaryotes) or eEF-2 (in eukaryotes) and the hydrolysis of GTP. The tRNA previously in the A site is moved to the P site.

• Antibiotic and Toxin Interference during Elongation:

  • Tetracyclines: Block the A site.

  • Clindamycin and Erythromycin: Bind irreversibly and inhibit translocase.

  • Diphtheria Toxin: Inactivates eEF-2, inhibiting eukaryotic elongation.

• Energy Cost: Each cycle of adding one amino acid requires the consumption of $4 \sim P$ (high-energy phosphate bonds):

  • $2 \sim P$ from ATP (hydrolyzed to $AMP + PP_i$) to attach the amino acid to the tRNA.

  • $1 \sim P$ from GTP (hydrolyzed to $GDP + P_i$) for EF-Tu function.

  • $1 \sim P$ from GTP (hydrolyzed to $GDP + P_i$) for EF-G function.

• Translation is therefore energetically expensive, which is a primary reason why cells require large amounts of GTP.

Termination of Translation

• Elongation concludes when one of the three stop codons enters the A site: UAA, UAG, or UGA. No tRNA corresponds to these codons.

• Releasing Factors (RFs) in Prokaryotes:

  • RF-1: Recognizes UAA and UAG.

  • RF-2: Recognizes UAA and UGA.

  • RF-3: Binds to GTP and stimulates the activity of RF-1 and RF-2.

• Releasing Factors in Eukaryotes:

  • Eukaryotes have only one releasing factor (eRF) that handles all stop codons and associated tasks.

• Mechanism of Termination:

  • Activated releasing factors cause the polypeptide to be released from the P site because there is no attachment point at the A site.

  • The peptide is released and the two ribosomal subunits dissociate.

  • Initiation factors reattach to the small subunit to begin the process again.

Specialized Translation Contexts

• Polysome: Defined as multiple ribosomes acting on a single strand of mRNA simultaneously to increase the rate of protein synthesis.

• Polycistronic mRNA: Common in prokaryotes, these mRNAs contain coding sequences for several different proteins. In these cases, ribosomes must initiate translation at specific, unique initiation points for each protein.