Ribosomes and protein synthesis lecture 2

BMS1025 – Cell BiologyInstructor: Dr. Penny LympanyContact: P.Lympany@surrey.ac.ukDate: April

Topics

Cellular Biology focusing on organelles, structure, function.

RNA Modification

Significance: RNAs undergo extensive modifications within the nucleus before exiting to ensure proper function and stability. These modifications include methylation and pseudouridylation, which affect RNA folding and translation efficiency.

Post-Translational Modification

Source: Conibear, A. C. (2020).Publication: Nature Reviews ChemistryFocus: Understanding protein post-translational modifications using chemical biology tools.These modifications, such as phosphorylation, glycosylation, and ubiquitination, play crucial roles in regulating protein activity, localization, and degradation, impacting cellular signaling pathways.

tRNA Processing

Key Points:

  • Synthesis: Eukaryotic tRNAs are synthesized by RNA polymerase III, which transcribes tRNA genes into precursors.

  • Precursors: tRNAs are synthesized as larger precursor tRNAs, which undergo several modifications, including 5' capping and 3' polyadenylation, to become mature forms.

  • Intron Splicing: Some precursor tRNAs contain introns that are spliced out through a catalytic process led by endonucleases and following ligation of the remaining exons.

  • Quality Control: Proper folding into a cloverleaf configuration is essential for tRNA functionality. tRNAs that fail to fold correctly are subjected to degradation by the nuclear exosome, ensuring only functional tRNAs are exported to the cytoplasm.

Editing Mechanisms in tRNA

Two Mechanisms:

  • Favorable Binding: The correct amino acid (AA) binds with the highest affinity to the active site of the tRNA synthetase, ensuring accuracy in translation.

  • Hydrolytic Editing: Synthetase attempts to insert adenylated AA into a second editing pocket that is designed to exclude the correct AA, thus enabling the removal of incorrectly charged AAs by hydrolytic activity.

Protein Synthesis - Elongation Process

Steps:

  1. Peptide Bond Formation: Catalyzed by the ribosomal peptidyl transferase activity, linking the amino acids together.

  2. Large Subunit Translocation: The large ribosomal subunit shifts, allowing elongation to proceed.

  3. Small Subunit Translocation: The small subunit moves to maintain proper interaction with the mRNA.Ribosome Movement: Moves 3 nucleotides along the mRNA, ensuring the correct sequence for translation.

Initiation of Protein Synthesis

Key Start Point: Begins at the AUG codon (Methionine), which is crucial to correctly establish the reading frame and prevent errors that can lead to non-functional proteins.

  • Initiator tRNA: Always carries modified formylmethionine (fMet) in bacteria or methionine in eukaryotes.

  • Complex Assembly: The initiator tRNA-methionine complex binds to the small ribosomal subunit along with various eukaryotic initiation factors (eIFs), facilitating the assembly.

  • Process: The small ribosomal subunit binds to the 5' end of mRNA and scans for the AUG codon. Upon recognition, initiation factors dissociate, allowing the large ribosomal subunit to assemble, positioning the initiator tRNA at the P site.

Termination of Protein Synthesis

Stop Codons: At the end of coding, the presence of one of three stop codons (UAA, UAG, UGA) signals termination of translation.Release Factors: These proteins bind to the ribosome at the A site, promoting the addition of a water molecule instead of an AA, which results in the release of the nascent polypeptide chain into the cytoplasm.Ribosome Disassembly: Following termination, the mRNA is released, and ribosomal subunits separate, preparing for a new translation cycle.

Polyribosomes in Protein Synthesis

Process: Multiple ribosomes can simultaneously initiate translation on the same mRNA transcript, significantly enhancing protein production, which can take from 20 seconds to several minutes, depending on the protein's complexity.Spacing: Ribosomes are approximately 80 nucleotides apart, effectively increasing translational efficiency.

Control Mechanisms in Protein Production

Cellular Safeguards:

  • Faulty mRNA: mRNAs that are broken or lack a 5' cap or poly A tail are prevented from engaging in initiation, maintaining integrity in protein synthesis.

  • Nonsense Mediated Decay: This mechanism ensures that mRNAs containing premature stop codons are degraded before they leave the nucleus.

Proteasomes

Function: Essential for the removal of unwanted or faulty proteins, which could otherwise accumulate and disrupt cellular processes.Mechanism: Proteins tagged for degradation are recognized and transported to the proteasome where they undergo proteolysis, ensuring normal cell function and homeostasis.

Structure of Proteasomes

Composition: Proteasomes are abundant in cells, comprising approximately 1% of total proteins, and are predominantly located in the cytosol and nucleus.

  • Degradation Process: Proteins are retrotranslocated to the cytosol for destruction by traveling through a central hollow cylinder composed of a six-subunit protein ring that facilitates substrate entry and unfolding.

Protein Folding into Functional Conformation

Requirements: Proper protein function requires achieving a correct three-dimensional conformation, binding to necessary cofactors, and undergoing numerous post-translational modifications to become active.

  • Folding Mechanism: Hydrophobic residues tend to aggregate in the core of the protein, while non-covalent bonds stabilize the resultant structure, leading to a stable functional form.

Role of Chaperones in Protein Folding

Definition: Molecular chaperones assist in the correct folding of proteins, preventing aggregation and misfolding that can result in cellular stress.

  • Heat-Shock Proteins (hsp): Their synthesis is elevated in response to various stress conditions, particularly temperature changes. Key families such as hsp60 and hsp70 operate in diverse cellular compartments to facilitate folding processes.

Hsp60 Chaperonins

Role: Specifically, certain proteins require Hsp60, which forms a barrel-shaped isolation chamber that provides a conducive environment for protein folding while preventing aggregation.

Regulated Protein Destruction

Why: Proteins are selectively eliminated due to factors such as misfolding, damage, or alterations in cell state, which could be detrimental to the organism.Ubiquitination: This marks target proteins for degradation by the proteasome through a complex series of regulatory mechanisms involving ubiquitin tags, which signal for destruction.

Summary of Protein Synthesis

Focus: The overall processes and various components involved in the intricate network of protein synthesis and regulation.

Signaling for Protein Destinations

Destinations: Proteins are directed to several different cellular locations, including secretion, plasma membrane integration, or delivery to lysosomes.Mechanism: Every newly synthesized protein begins in the endoplasmic reticulum (ER); those designated for mitochondria, chloroplasts, or the nucleus utilize distinct pathways. Signal sequences are instrumental in directing proteins to their final locations and are subsequently removed post-transport.

Signal Recognition Particle (SRP)

Function: Recognizes specific signal sequences within nascent proteins and facilitates the transfer of the ribosome-polypeptide complex to the ER, coordinating the import of new proteins into the secretory pathway.

Glycosylation Process

Process: Proteins undergoing glycosylation in the ER are modified and sorted for various destinations, including further processing in the Golgi complex, which plays a key role in determining protein function and cellular communication.

N-Linked Glycosylation

Process: This critical step involves the addition of oligosaccharides to asparagine residues in proteins, which is vital for proper folding, stability, and function.

Glycosylation Steps

Events: Initial steps of N-linked glycosylation occur on the cytosolic face of the ER, with further modifications continuing as proteins are transported through the Golgi complex, which is essential for the final maturation and sorting of glycoproteins.

Conclusion

Further Information: For additional resources and detailed discussions, refer to the textbook "Molecular Biology of the Cell" or the course discussion board for insights and expanded learning opportunities.

Glossary of Key Terms

  • Aminoacyl-tRNA Synthetase: Enzyme that attaches the correct amino acid to its corresponding tRNA, ensuring fidelity in translation.

  • Anticodon: Sequence in tRNA that pairs with the complementary mRNA codons, crucial for the accuracy of protein synthesis.

  • Codon: A three-nucleotide sequence in mRNA that specifies a particular amino acid during translation.

  • Proteasome: A complex responsible for degrading unneeded or damaged proteins, maintaining cellular health.

  • Ribosome: Composed of ribosomal RNA (rRNA) and proteins, ribosomes are the molecular machines responsible for protein synthesis.

  • Ribozyme: An RNA molecule that possesses catalytic activity, playing roles in biological reactions, including RNA splicing.

  • Transfer RNA (tRNA): Small RNA molecules that are key to translating mRNA codons into the corresponding amino acids during protein synthesis.