Protein Synthesis and Turnover

Protein Synthesis and Turnover

Protein Synthesis

Translation Overview

  • Translation Process: Protein translation occurs in three main steps:

    • Initiation:

    • The start codon (AUG) must be located.

    • Elongation:

    • Codons are read sequentially from 5′ to 3′.

    • Proteins are synthesized from the amino end to the carboxyl end.

    • Termination:

    • Stop codon is read.

    • Polypeptide is hydrolyzed from the last tRNA.

    • The translation machinery comes apart, facilitating another round of translation.

Initiation: Decoding mRNA

  • Ribosome Structure: Ribosomes have three sequential tRNA binding sites that allow the sequential addition of amino acids encoded by mRNA.

  • mRNA Interaction: mRNA interacts with the 30S ribosomal subunit.

  • tRNA Binding Sites:

    • Sites include:

    • A Site (Aminoacyl Site)

    • P Site (Peptidyl Site)

    • E Site (Exit Site)

  • Peptide Bond Formation: The acceptor ends of the tRNA at the A and P site converge, creating a site for peptide bond formation through a tunnel that allows the polypeptide chain to pass.

Initiation: Translation Start Signal

  • Start Codon: The codon AUG specifies Methionine and begins the polypeptide chain.

  • Encoding Signals: All known mRNA molecules contain signals that define the beginning and end of each encoding polypeptide chain.

  • Upstream Nucleotides: These purine-rich sequences, known as Shine-Dalgarno sequences, facilitate mRNA binding to rRNA within the 30S subunit.

  • Pairings for Synthesis Start in Bacteria:

    • mRNA to the 16S rRNA

    • Start codon on mRNA to the complementary anticodon on tRNA

Initiation: Methionine Starts Synthesis

  • In bacteria, Methionine is modified with a formyl group, referred to as fMet.

  • The formyl group is removed in some proteins once the polypeptide chain reaches about 10 amino acids.

  • The initiator tRNA is charged with fMet, indicating a two-step process in initiation.

Formation of the 70S Initiation Complex

  • Initiation Factors:

    • IF1, IF2, IF3 assemble the 30S subunit, which pairs with the 50S subunit to form the 70S initiation complex.

    • Binding Process:

    • IF1 and IF3 bind to the 30S subunit.

    • IF3 brings fMet-tRNA.

    • The combination of IF1, IF3, and fMet-tRNA at the P site forms the 30S complex.

    • IF2 stimulates the binding of the 50S subunit, resulting in the 70S initiation complex.

  • Ribosome State: The ribosome is established and is now ready for the elongation phase, with A and E sites empty, establishing the reading frame for translation.

Elongation Factors Deliver Aminoacyl-tRNAs

  • Function of EF-Tu:

    • Delivers the next aminoacyl-tRNA to the A site.

    • Protects aminoacyl-tRNA from hydrolysis.

    • Contributes to the accuracy of protein synthesis.

Conformational Change Induced by EF-Tu

  • Interaction with the 16S subunit: EF-Tu (GTP) interacts with the 16S subunit, facilitating a conformational change that positions aminoacyl-tRNAs at the P and A sites, thus promoting peptide bond formation—a process known as accommodation.

  • Release Mechanism: EF-Tu (GDP) is released, and EF-Ts then induces the release of GDP from EF-Tu, resetting the cycle for the next aminoacyl-tRNA.

Peptide Bond Synthesis

  • Bond Formation:

    • The P and A sites must be occupied for peptide bond formation.

    • This formation is thermodynamically spontaneous and occurs in the 50S subunit at the peptidyl transferase center.

    • It is catalyzed by a site on the 23S rRNA.

  • Catalytic Mechanism: Catalysis occurs through proximity and orientation, positioning substrates to allow spontaneous formation of peptide bonds.

Translocation of tRNAs and mRNA

  • Cycle Process:

    • The peptidyl-tRNA occupies the A site while the aminoacyl-tRNA binds the P site.

    • A peptide bond is formed.

    • tRNAs and mRNA translocate via EF-G, moving the deacylated tRNA to the E site.

    • The movement is by one codon and is induced by a conformational change.

  • tRNA Dissociation: After translocation, tRNA is ready to dissociate, completing the cycle.

Polypeptide Chain Growth

  • Process: The peptide chains remain in the P site, expanding through the exit tunnel as the elongation cycle repeats with new aminoacyl-tRNAs entering the A site.

  • Frequency of Synthesis: Takes place approximately 40 times per second.

  • Directionality: Synthesis proceeds from the amino (N-) terminal to the carboxyl (C-) terminal direction, and multiple ribosomes can translate simultaneously.

Synthesis Termination

  • Role of Release Factors: Termination is mediated by release factors (RF) that recognize stop codons.

  • Binding Mechanism: When bound, RF bridges the stop codon on mRNA with the peptidyl transferase center on the 50S subunit, resulting in the detachment of the polypeptide from the ribosome.

  • Complex Dissociation: tRNA and mRNA remain attached to the 70S ribosome briefly until complex dissociation occurs, driven by GTP hydrolysis in response to EF-G and ribosome release factor (RRF).

Bacteria vs Eukaryote Protein Synthesis

  • Size of Ribosomes: Eukaryotic ribosomes are larger, comprising a 60S and a 40S subunit forming an 80S complex.

  • Initiator tRNA Differences: In eukaryotes, the initiator tRNA is Methionine rather than N-formylmethionine as in bacteria.

Eukaryote Initiation Process

  • Complex Assembly: Eukaryotic initiation begins with the assembly of a complex on the 5′ cap involving Met-tRNA, the 40S ribosome, and initiation factor eIF-2.

  • Scans for Start Codon: Driven by ATP hydrolysis, the complex scans mRNA until it locates the first start codon (AUG).

  • Complex Formation: Upon encounter with a start codon, the 60S subunit is added to form the 80S initiation complex.

Additional Differences

  • mRNA Structure: Eukaryotic mRNA is circular, with a 5′ cap attached to a poly A tail, though the benefit of this structure is unknown.

  • Termination Differences: In eukaryotes, termination requires only one release factor.

  • Efficiency Factors: The organization of translation complexes in eukaryotes is associated with the cytoskeleton, enhancing protein synthesis efficiency.

Protein Sorting in Eukaryotes

  • Mechanisms of Delivery: Eukaryotes employ two mechanisms to direct newly synthesized proteins to their correct locations.

    • Cytoplasmic Proteins: These proteins are correctly positioned upon synthesis within the cytoplasm.

    • Mitochondrial and Nuclear Proteins: Some proteins are synthesized in the cytoplasm but delivered post-translation.

Secretory Pathway

  • Translation in ER: Proteins intended for secretion, lysosomes, Golgi complex, and membrane proteins are synthesized in the endoplasmic reticulum (ER).

  • Ribosome Behavior: Ribosomes in eukaryotes can be free in the cytoplasm unless directed to the rough ER where synthesis continues in the ER lumen.

Free Cytoplasmic Ribosomes

  • Synthesis Process: Protein synthesis begins in the cytoplasm but is temporarily halted until the ribosome is directed to the ER.

  • Docking and Resuming Synthesis: Once the ribosome docks onto the ER, synthesis resumes, directing the nascent polypeptide chain into the ER lumen.

  • Identical Ribosomes: Free ribosomes synthesizing cytoplasmic proteins are identical to those bound to the ER.

The Four Components of the Secretory Pathway

  • Signal Sequence:

    • Comprises 9 to 12 hydrophobic amino acids mixed with some positively charged amino acids.

    • Functions to identify the protein for ER membrane crossing.

  • Signal-Recognition Particle (SRP):

    • Binds the signal sequence, halting protein synthesis.

    • Translocates the ribosome and the nascent polypeptide to the ER.

  • GTPase Functionality:

    • The SRP Receptor, which is also a GTPase, binds the SRP to facilitate further processing.

  • Translocon Role:

    • This protein-conducting channel opens when the SRP-SRP receptor complex binds, allowing protein synthesis to resume.

Protein Synthesis Regulation

  • Control Mechanisms: Protein synthesis is controlled by a variety of mechanisms:

    • The availability of mRNA, ensuring that proteins are not translated unless there is a cellular requirement.

    • Stability of mRNA, where certain elements can be bound by proteins for stabilization and subsequent translation.

    • Small non-coding RNAs regulate mRNA stability and usage.

    • miRNA signals presence for mRNA translation or cleavage.

Summary of Key Concepts

  • The genetic code establishes the connection between nucleic acids and proteins.

  • Amino acids are activated upon attachment to tRNA, which provides the necessary components to form proteins.

  • Ribosomes function as ribonucleoproteins composed of two significant subunits.

  • Protein synthesis involves the decoding of mRNA, with peptidyl transferase catalyzing the formation of peptide bonds.

  • Bacterial and eukaryotic protein synthesis processes share similarities but also have crucial differences that affect functionality.

  • Ribosomes that are bound to the ER synthesize proteins intended for secretion or integration into membranes.

  • Various mechanisms exist to meticulously regulate protein synthesis, ensuring cellular efficiency and need-based response.