Mechanism of Translation II: Elongation and Termination

Synthesis Directionality: Proteins are synthesized in the amino-to-carboxyl ( $N ightarrow C$ ) direction. * This implies that the amino-terminal ( $N$ -terminal) amino acid is the first to be incorporated into the growing polypeptide chain.
mRNA Reading Directionality: Ribosomes read Messenger RNAs ( $mRNAs$ ) in the $5' ightarrow 3'$ direction. * This is functionally significant as it is the same direction in which $mRNAs$ are synthesized by $RNA$ polymerase.
Fundamental Comparison: While elongation is highly conserved, showing significant similarities between bacteria and eukaryotes, there are specific differences in factors and mechanisms that define each domain.

Definition of the Genetic Code: The genetic code refers to the set of 3-base code words, known as codons, in $mRNA$ that represent the $20$ standard amino acids found in proteins.
The Triplet Code: Codons consist of three-base sequences. These instruct the ribosome to incorporate specific amino acids into a polypeptide.
Non-overlapping Nature: The code is strictly non-overlapping, meaning each nucleotide base in the sequence is a part of only one specific codon.
Lack of Gaps or Commas: The code is devoid of gaps or "commas." Every base within the coding region of an $mRNA$ is accounted for as part of a codon, ensuring a continuous reading frame.

Degeneracy of the Code: The genetic code is degenerate, meaning multiple codons can code for the same amino acid. This is accommodated by two primary mechanisms: * Isoaccepting Species of tRNA: These are different $tRNA$ molecules that bind the same amino acid but recognize different codons. * The Wobble Hypothesis: The base at the $3^{rd}$ position of a codon is permitted to move slightly from its standard position. This flexibility allows it to form non-Watson-Crick base pairs with the anticodon of a $tRNA$ .
Wobble Base Pairs: The specific unusual base pairs allowed by wobble include: * $G ext{-}U$ base pairing. * $I ext{-}A$ base pairing (where $I$ represents Inosine).
Functional Implication: Wobble allows a single aminoacyl- $tRNA$ species to pair with more than one codon, reducing the total number of $tRNAs$ required by the cell.

Non-universality: While once thought to be strictly universal, the genetic code is now known to have exceptions.
Observed Alterations: * Eukaryotic Nuclei and Mitochondria: Certain nuclei and mitochondria deviate from the standard code. * Bacteria: At least one bacterium has been identified with an altered code.
Common Deviations: * Codons that are normally termination signals (stop codons) in the standard code can be reassigned to code for amino acids such as Tryptophan ( $Trp$ ) or Glutamic Acid ( $Glu$ ). * In mitochondrial genomes and the nuclei of at least one species of yeast, the sense of a codon can be changed from one amino acid to another.
Evolutionary Context: Deviant codes remain closely related to the standard code, indicating they evolved from it. This raises the question of whether the code is a "frozen accident" or a product of evolution shaped by the ability to cope with mutations.

Step 1: Aminoacyl-tRNA Binding: Elongation factor $EF ext{-}Tu$ brings the second aminoacyl- $tRNA$ to the ribosomal $A$ site.
Step 2: Peptide Bond Formation: The enzyme peptidyl transferase forms a peptide bond between the initiator $fMet$ (or the existing peptide) and the new aminoacyl- $tRNA$ . * This lengthens the peptide by one amino acid. * The elongated peptide is shifted to the $A$ site.
Step 3: Translocation: Elongation factor $EF ext{-}G$ shifts the $mRNA$ and the $tRNAs$ one codon's width to the left. * This moves the dipeptidyl- $tRNA$ into the $P$ site. * The deacetylated (deacylated) $tRNA$ moves to the $E$ (Exit) site. * The $A$ site is opened to receive the next aminoacyl- $tRNA$ .

EF-T (Transfer): This factor is responsible for transferring aminoacyl- $tRNAs$ to the ribosome. It is a complex composed of two distinct proteins: * EF-Tu: The "u" stands for thermo-unstable. It is involved in the first step of elongation. * EF-Ts: The "s" stands for thermo-stable. It facilitates the regeneration of $EF ext{-}Tu ext{-}GTP$ .
EF-G: The "G" stands for $GTPase$ activity. This factor is essential for the third step of elongation (translocation).

Binary Complex Formation: $EF ext{-}Tu$ couples with $GTP$ to form a binary complex.
Ternary Complex Formation: This complex associates with an aminoacyl- $tRNA$ to form a ternary complex ( $EF ext{-}Tu ext{-}GTP ext{-}aa ext{-}tRNA$ ).
Ribosome Binding: The ternary complex binds to the ribosome at the $A$ site.
Hydrolysis and Dissociation: $GTP$ is hydrolyzed into $GDP$ . The resulting $EF ext{-}Tu ext{-}GDP$ complex dissociates, leaving the aminoacyl- $tRNA$ positioned in the $A$ site.
Recycling: $EF ext{-}Ts$ exchanges $GTP$ for $GDP$ on $EF ext{-}Tu$ , regenerating the active $EF ext{-}Tu ext{-}GTP$ binary complex for the next round.

Peptidyl Transferase Activity: Once initiation factors and $EF ext{-}Tu$ have completed their roles, the ribosome contains $fMet ext{-}tRNA$ in the $P$ site and an aminoacyl- $tRNA$ in the $A$ site. * The ribosome itself contains the enzymatic activity (peptidyl transferase) required to form the peptide bond. * No new elongation factors are required specifically for the bond formation itself.
Translocation Mechanics: After bond formation, the ribosome has a peptidyl- $tRNA$ in the $A$ site and a deacylated $tRNA$ in the $P$ site. * Translocation moves the $mRNA$ and peptidyl- $tRNA$ exactly one codon's length through the ribosome. * This requires $EF ext{-}G$ and $GTP$ hydrolysis. * Release of $EF ext{-}G$ from the ribosome is dependent on $GTP$ hydrolysis, which is necessary for a new round of elongation to occur.

Termination Signals: Translation stops when the ribosome encounters a stop codon.
Natural Stop Codons: * $UAG$ * $UAA$ * $UGA$
Premature Termination Mutations: Mutations that create stop codons within an $mRNA$ sequence can end translation early: * Amber Mutation: Creates a $UAG$ codon. * Ochre Mutation: Creates a $UAA$ codon. * Opal Mutation: Creates a $UGA$ codon.

Prokaryotic Termination Factors: Three factors mediate the process: * RF1: Recognizes stop codons $UAA$ and $UAG$ . * RF2: Recognizes stop codons $UAA$ and $UGA$ . * RF3: A $GTP$ -binding protein that facilitates the binding of $RF1$ and $RF2$ to the ribosome.
Ribosome Release (Prokaryotes): Ribosomes do not release from $mRNA$ spontaneously. * RRF (Ribosome Recycling Factor): Resembles a $tRNA$ in structure. It binds to the ribosomal $A$ site in a position not normally occupied by $tRNA$ . * Collaboration: Working with $EF ext{-}G$ , $RRF$ facilitates the release of either the $50S$ ribosome subunit or the entire ribosome structure.
Eukaryotic Termination Factors: * eRF1: A single factor that recognizes all three termination codons ( $UAG, UAA, UGA$ ). * eRF3: A ribosome-dependent $GTPase$ that assists $eRF1$ in releasing the completed polypeptide. * Ribosome Release: Eukaryotic ribosomes are released by $eIF3$ , aided by additional factors $eIF1$ , $eIFA$ , and $eIF3j$ .