Translation Mechanisms, Mutations, and Neuronal Signaling: Structured Study Notes for Cell Biology

Translation Mechanisms and Factors

Elongation in Protein Synthesis

Elongation is a key phase of translation during which the polypeptide chain is extended by the sequential addition of amino acids. In bacteria, elongation involves a repetitive cycle of three main steps:

• Binding of aminoacyl tRNA: An aminoacyl tRNA is brought to the ribosome, positioning a new amino acid for addition to the growing peptide chain.

• Peptide bond formation: The amino acid is covalently linked to the polypeptide via a peptide bond.

• Translocation: The mRNA is shifted three nucleotides, bringing the next codon into position for translation.

Additional info: Eukaryotic elongation mechanisms are more complex and involve additional factors, but this section focuses on bacterial systems.

Elongation Factors and GTP Hydrolysis

• EF-Tu-GTP complex: Delivers aminoacyl tRNA to the A site of the ribosome.

• GTP hydrolysis: Occurs as the aminoacyl tRNA is transferred, releasing EF-Tu-GDP.

• EF-Ts: Regenerates EF-Tu-GDP to EF-Tu-GTP, allowing the cycle to continue.

Binding of tRNA

• All tRNAs (except initiator tRNAs) are brought to the A site.

• Only tRNAs with anticodons complementary to the codon remain at the A site for GTP hydrolysis.

• The final error rate in translation is very low, approximately 1 in 10,000.

Peptide Bond Formation

• A peptide bond forms between the amino group of the amino acid at the A site and the carboxyl group of the amino acid at the P site.

• The growing peptide chain is transferred to the tRNA at the A site.

• No ATP or GTP hydrolysis is required for this step.

Role of rRNA in Peptide Bond Formation

• 23S rRNA in bacteria contains the peptidyl transferase activity, acting as a ribozyme.

• This demonstrates that RNA can have catalytic functions, not just informational roles.

Translocation

• After peptide bond formation, the mRNA advances to bring the next codon into position.

• Peptidyl tRNA moves from the A site to the P site; empty tRNA moves to the E site.

• Hydrolysis of GTP bound to EF-G triggers conformational changes that complete these movements.

Termination of Polypeptide Synthesis

• Translation continues until a stop codon arrives at the A site.

• Stop codons are recognized by release factors (proteins), not tRNAs.

• Release factors have regions that bind mRNA stop codons at the A site, mimicking tRNA structure (molecular mimicry).

• Binding of release factors (with GTP) triggers release of the polypeptide from the ribosome.

Types of Mutations and Their Effects on Translation

Base-Pair Substitutions

• Mutation: Any change in the nucleotide sequence of a genome.

• Missense (nonsynonymous) mutation: Alters a codon to encode a different amino acid (e.g., sickle-cell anemia: GUA replaces GAA, valine replaces glutamic acid).

Nonstop and Nonsense Mutations

• Nonstop mutation: Changes a stop codon to an amino acid codon, resulting in continued translation.

• Nonsense mutation: Changes an amino acid codon to a stop codon, causing premature termination and a truncated polypeptide.

• Special names for nonsense mutations:

  • Amber mutation – premature UAG

  • Ochre mutation – premature UAA

  • Opal/umber mutation – premature UGA

Frameshift Mutations

• Result from base-pair insertions or deletions (indels).

• Cause a shift in the reading frame, potentially generating nonsense, nonstop, or missense codons.

Other Types of Mutations

• Some amino acid substitutions may not affect protein function if the substituted amino acids are similar.

• Silent (synonymous) mutations: Affect the third base of the codon but do not change the encoded amino acid.

Nonsense-Mediated Decay and Nonstop Decay

• Nonsense-mediated decay: Eukaryotic cells destroy mRNAs with premature stop codons to prevent production of incomplete proteins.

• Exon junction complex (EJC): Multiprotein complex deposited at exon-exon junctions during splicing; used to detect premature stop codons.

• If a stop codon is present before the final EJC, translation is terminated and the mRNA is targeted for degradation.

Fate of mRNAs with No Stop Codon

• Translation stalls if a ribosome encounters an mRNA lacking a stop codon.

• In eukaryotes, RNA-degrading enzymes (involving Ski7p and exosome proteins) bind the empty A site and degrade the defective mRNA (nonstop decay).

• In bacteria, transfer messenger RNA (tmRNA) binds the A site, allowing translation to terminate and tagging the defective protein for degradation.

tmRNA

• tmRNA is a unique RNA with both tRNA and mRNA domains.

• The tRNA domain is charged with an amino acid; the mRNA domain contains a stop codon and codes a tag sequence for protease targeting.

Posttranslational Processing

Posttranslational Modification of Polypeptides

After synthesis, polypeptide chains often undergo modifications to become functional proteins.

• In bacteria, the N-formyl group is always removed; the methionine at the N-terminus is often removed.

• In eukaryotes, the methionine at the N-terminus is often released as well.

Nervous Tissue and Neuronal Structure

Overview of the Nervous System

The nervous system transmits impulses along specialized plasma membranes of nerve cells. Vertebrates have:

• Central nervous system (CNS): Brain and spinal cord.

• Peripheral nervous system (PNS): Sensory and motor components outside the CNS.

Cells of the Nervous System

• Neurons: Send and receive electrical impulses.

  • Sensory neurons: Detect stimuli.

  • Motor neurons: Transmit signals from CNS to muscles/glands.

  • Interneurons: Transmit information within the nervous system.

• Glial cells: Support neurons; most abundant in CNS.

  • Microglia: Fight infection and remove debris.

  • Oligodendrocytes (CNS) and Schwann cells (PNS): Form myelin sheath.

  • Astrocytes: Control blood-brain barrier.

Structure of Neurons

• Cell body: Contains nucleus and endomembrane components.

• Dendrites: Receive signals.

• Axons: Conduct signals; may be surrounded by myelin sheath.

Axons and Myelin Sheath

• Myelin sheath insulates axons, formed by oligodendrocytes (CNS) and Schwann cells (PNS).

• Nodes of Ranvier are gaps between myelinated segments where action potentials are renewed.

• Myelination decreases membrane capacitance, allowing faster signal transmission.

Motor Neurons and Synaptic Boutons

• Motor neurons have multiple dendrites and a single long axon.

• Axon branches terminate in synaptic boutons, transmitting signals to the next cell.

Synapses

• A synapse is the junction between a nerve cell and another cell (neuron, muscle, or gland).

• Synaptic terminals release neurotransmitters to transmit signals across the synapse.

• The presynaptic cell sends the signal; the postsynaptic cell receives it.

• Signal transmission is unidirectional.

Membrane Potential and Its Maintenance

Resting Membrane Potential

All cells have a voltage difference across their plasma membrane, called the membrane potential. In neurons, the resting potential is typically about -60 mV (e.g., squid giant axon).

• Cells at rest have excess negative charge inside and positive charge outside.

• Resting potential is maintained by ionic gradients and selective permeability.

Role of Na+/K+ Pump

• The Na+/K+ pump actively transports three sodium ions out and two potassium ions in, maintaining the potassium gradient.

• This pump is essential for maintaining the resting membrane potential.

• Equation for the pump's stoichiometry: $3\,\text{Na}^+_{\text{out}} : 2\,\text{K}^+_{\text{in}}$

Potassium Leak Channels

• Potassium leak channels allow K+ to diffuse out, leaving behind anions and creating a negative resting potential.

Action Potential

• Action potentials are rapid changes in membrane potential triggered by stimuli.

• Membrane potential shifts from negative to positive and back in a short time.

• Voltage-gated channels (Na+, K+) open/close in response to voltage changes, generating current (measured in amperes).

Patch Clamping and Ion Channel Study

• Patch clamping allows recording of ion currents through individual channels.

• Developed by Erwin Neher and Bert Sakmann.

Voltage-Gated Channel Structure and Function

• Channels are multimeric proteins (e.g., sodium channels are large monomers with four domains).

• Each domain/subunit contains six transmembrane segments.

• Channel specificity is determined by the size and chemical environment of the central pore.

• Channel gating is all-or-none; channels are either open or closed.

• Helix S4 acts as a voltage sensor.

• Channels can undergo inactivation, where an inactivating particle blocks the pore and prevents reopening.

Table: Types of Mutations and Their Effects

Mutation Type	Genetic Change	Effect on Protein	Example
Missense	Base-pair substitution	Wrong amino acid incorporated	Sickle-cell anemia
Nonsense	Base-pair substitution	Premature stop codon, truncated protein	Amber (UAG), Ochre (UAA), Opal (UGA)
Nonstop	Base-pair substitution	Loss of stop codon, extended protein	Nonstop mRNA decay
Frameshift	Insertion/deletion (indel)	Altered reading frame, multiple wrong amino acids	Genetic diseases, defective proteins
Silent	Base-pair substitution (third codon position)	No change in amino acid	Synonymous codon usage

Example: Sickle-Cell Anemia

• Mutation: AT base pair substituted for TA in DNA.

• mRNA codon changes from GAA to GUA.

• Protein: Valine replaces glutamic acid, altering hemoglobin function.

Additional info:

• Spliceosome components (U1, U2, U4/U6, U5) are essential for pre-mRNA splicing, but detailed roles are not covered in these slides.

• Antibiotics targeting translation (e.g., tetracycline, chloramphenicol) bind specific ribosomal sites and inhibit protein synthesis.