mRNA Transport and Protein Translation

Protein Synthesis

• mRNA transports out of the nucleus to be translated.

mRNA Transport Out of the Nucleus

• A 5' cap and a poly-A tail protect mRNA from degradation and assist in ribosome binding.
• The 5' cap is 7-methylguanosine.
• The poly-A tail stabilizes mRNA, aids in nuclear export, and influences translation efficiency.
• Nuclear pore complexes (NPCs) are large protein structures regulating the transport of molecules between the nucleus and cytoplasm.
• NPCs facilitate selective transport of export-ready mRNA.
• Export-ready mRNA must be properly processed, with 5' capping, splicing, and polyadenylation.
• Export-ready mRNA associates with exon-junction complexes (EJCs), poly-A binding proteins, and nuclear export receptors.

Protein Translation

• mRNA codons (triplets of nucleotides) specify amino acids via tRNAs, not single nucleotides directly.
• Codons comprise three consecutive nucleotides.
• The genetic code is redundant, allowing multiple codons to specify the same amino acid.
• There are 64 total codons, with 61 for amino acids and 3 stop codons.
• tRNA serves as an adaptor molecule, linking mRNA codons to specific amino acids through its anticodon.
• The anticodon is a sequence of three nucleotides on tRNA that base-pairs with mRNA codons.
• Aminoacyl-tRNA synthetase attaches the correct amino acid to tRNA.
• Ribosomes are located in the cytoplasm or on the rough ER for secretory/membrane proteins.
• Ribosomes are assembled in the nucleolus where rRNA genes are found in nucleolar organizer regions.
• Peptidyl transferase (a ribozyme) catalyzes peptide bond formation.
• The mRNA-binding site is located in the small ribosomal subunit.
• The three ribosomal sites include:
  • The A site which Accepts incoming aminoacyl-tRNA.
  • The P site which Holds tRNA with growing polypeptide.
  • The E site which is Exit site for empty tRNA.
• During translation, polypeptide chain growth has new amino acids added to the carboxyl end.
• Peptide bond formation is energetically favorable due to ribosome-assisted catalysis.
• The ratcheting mechanism involves elongation factors and GTP hydrolysis.
• Initiator tRNA carries methionine, which is the AUG start codon.
• The small ribosomal subunit binding site recognizes the start codon with the initiator tRNA.
• Translation terminates when release factors recognize stop codons (UAA, UAG, UGA) and disassemble the ribosome.

Protein Folding and Degradation

• Some proteins fold during synthesis with assistance from chaperones, known as co-translational folding.
• Misfolded proteins are degraded in degradation pathways.
  • Proteasome: A complex that degrades misfolded proteins.
  • Proteolysis: The breakdown of proteins into peptides/amino acids.
  • Ubiquitin Role: Tags proteins for degradation (polyubiquitination).

Key Concepts for Protein Synthesis

• The genetic code is redundant, universal, and read in triplets (codons).
• mRNA must leave the nucleus via nuclear pores before translation.
• tRNA & Aminoacyl-tRNA Synthetase ensures correct amino acid attachment.
• Ribosomes have large & small subunits made of rRNA + proteins.
• Ribosome binding sites:
  • A site (Aminoacyl) holds incoming tRNA.
  • P site (Peptidyl) holds a growing polypeptide chain.
  • E site (Exit) is the tRNA exit from ribosome.
• A large ribosomal subunit catalyzes peptide bond formation by the enzyme peptidyl transferase.
• Proteins tagged with ubiquitin are degraded by proteasomes.

Membrane Structure and Function

• Membranes separate internal and external environments and control substance movement.
• Selective Transport controls the movement of substances in and out of the cell
• Signal Transduction allow receptors to detect and respond to extracellular signals.
• Cell membranes enable Cell Recognition and Interaction which is important for immune responses and tissue formation.
• Energy Transformation is essential in processes like ATP synthesis in mitochondria.

Major Membrane Lipids

• Phospholipids are composed of a glycerol backbone, two fatty acid tails, and a phosphate-containing head.
  • Serve as primary components of membranes and form bilayers due to the amphipathic nature.
• Shorter fatty acid tails increases membrane fluidity.
• Unsaturated fatty acid tails (double bonds) increases membrane fluidity.
• Saturated fatty acid tails (no double bonds) decreases membrane fluidity.
• Sphingolipids provide structural stability and are involved in cell signaling.
  • They are derived from sphingosine instead of glycerol.
  • They're found in nervous system tissues like the myelin sheath.
• Sphingomyelin is a type of sphingolipid found in myelin.
• Tay-Sachs Disease is a genetic disorder caused by a deficiency in an enzyme that degrades sphingolipids, leading to neurodegeneration. Functions: Play a role in cell recognition and signaling.

Glycolipids

• Glycolipd's function is to play a role in cell recognition and signaling.
• Glycosphingolipids are its most common type in animal cells.
• Carbohydrates are located on the extracellular surface of the plasma membrane.
• Glycosylation occurs in the golgi apparatus.

Sterols

• Sterols function to regulate membrane fluidity and stability.
• Cholesterol is the primary sterol found in animal cells.
• At high temperatures cholesterol reduces fluidity by stabilizing the membrane.
• At low temperatures cholesterol prevents solidification by disrupting lipid packing.

Membrane Assembly and Maintenance

• The amphipathic nature of phospholipids leads to spontaneous bilayer formation due to hydrophobic and hydrophilic interactions.
• Enzymes Inserting New Phospholipids: Scramblases (random distribution) and flippases/floppases (asymmetric distribution).
• Lipid and Protein Distribution in Bilayers: Lipid composition varies between cytoplasmic and extracellular leaflets.
• Microdomains enriched in cholesterol and sphingolipids are known as lipid rafts and are involved in signaling and trafficking.

Membrane Proteins

• Integral membrane proteins are embedded in hydrophobic regions and are hard to extract without detergents.
  • Transmembrane proteins span the membrane.
  • Membrane-associated proteins are located on the cytosolic face.
  • Lipid-linked proteins are covalently attached to lipids.
• Peripheral membrane proteins are surface-bound and interacts with membrane lipids or integral proteins, and are located on both extra- and intra-cellular faces.
  • They are easier to extract than integral proteins.
• Glycoproteins and Proteoglycans which contain sugars attached to proteins, are found on the extracellular side of membranes, and are involved in recognition and adhesion.
• The membrane is not rigid but instead dynamic and fluid.
• Hydrophobic interactions and van der Waals forces hold lipid molecules together.
  • There are four types of Lipids:
    • Lateral Diffusion: Lipids move side-to-side within a leaflet.
    • Flexion: Fatty acid tails wiggle.
    • Rotation: Individual lipids spin in place.
    • Flip-Flop (Rare): Lipids move between leaflets, and requires enzymes

Restricted Movement of Membrane Proteins

• Barriers created by tight junctions restrict movement.
• Cytoskeletal anchors attach proteins to cytoskeleton.
• Extracellular matrix interactions restrict mobility.
• Short and unsaturated fatty acid chains increase fluidity.
• Cholesterol content acts as a buffer for fluidity.
• Higher temperatures increase fluidity, while lower temperatures decrease it.
• Spectrin Proteins provide structural support to the membrane, maintaining cell shape.

Membrane Transport

• Membrane transport is maintaining homeostasis, nutrient exchange and cell signalling.
• Simple diffusion has no energy input. - Small nonpolar molecules (O2, CO2, N2) - Hydrophobic molecules (steroid hormones) - Small uncharged polar molecules (H2O, ethanol) - Large uncharged polar molecules (glucose) - Charged molecules/ions (Na+, K+, Cl-) - Impact of hydrophobic membrane interior: The lipid bilayer prevents the passage of polar or charged molecules but allows hydrophobic molecules to pass freely.

Gradients in Membrane Transport

• Simple and passive diffusion is driven by two gradients: - Concentration Gradient: Difference in solute concentration across the membrane. - Electrochemical Gradient: Combined effect of concentration gradient and membrane potential (voltage).
• The driving force for ion movement dictates whether ions move into or out of the cell.
• The electrochemical gradient in coupled transport is used to drive transport of another molecule against its gradient. - Ex: Na*-glucose symporter

Transport Proteins: Channels vs Transporters

• Passive transport requires a membrane protein but does not require energy. - Molecules move down their gradient.
• Uniports transport a molecule in one direction, GLUT1 is an example.
• The 5 types of Transmembrane Channel Proteins include: - Porins: Large, non-specific channels for small molecules. - Aquaporins: Specialized channels for water transport. - Ion channels: Selective for specific ions. - Gap junctions: Allow communication between adjacent cells. - Gated channels: Open/close in response to stimuli.
• Ion channels are selective for specific ions, determined by the pore size and charge of amino acids lining the channel. - They are found in nerve, muscle, and epithelial cells. - Transport is very fast.
• Gated ion channels open and close in response to specific stimuli, voltage and ligand.
• Patch clamping measures current flow through individual ion channels to study behavior.
• Active transport requires energy (ATP or ion gradients) to move molecules against their concentration gradient. - Types include: - Coupled transporters: One molecule's gradient energy to transport another molecule. - Membrane pumps: ATP hydrolysis to pump molecules against their gradient.

Active Transport

• Symports: Two molecules move in the same direction e.g Na+/glucose
• Antiports: Move two molecules in opposite directions. Example: Na+/Ca2+ exchanger (moves Na* in, Ca²+ out).

ATPase Pumps

• Has 4 different types: P, F, V, and ABC

How Cells Obtain Energy from Food

Cells break down sugars, fatty acids, and amino acids through oxidation reactions to obtain energy; the released energy is stored in a chemical form as ATP.

• Cells obtain energy by breaking down sugars, fats, and amino acids through oxidation reactions.The energy released is stored in activated carrier molecules like ATP (adenosine triphosphate), NADH (nicotinamide adenine dinucleotide), and FADH2 (flavin adenine dinucleotide).
• Reaction Favorability determines the direction of a reaction.
  • Exergonic reactions (energy-releasing) are spontaneous.
  • Endergonic reactions (energy-requiring) are non-spontaneous.
• Unfavorable reactions (AG > 0) occur paired with favorable reactions (AG < 0) to ensure an overall negative AG.

ATP Production in Animal Cells

• Direct ATP synthesis occurs in glycolysis and the citric acid cycle.
• An example is ATP formation in glycolysis Step 10.
• Oxidative Phosphorylation: (Major ATP source)
  • Occurs in mitochondria.
  • It uses energy from NADH and FADH2 to generate ATP.

Glycolysis and Key Steps

• Cellular Location: Cytoplasm
• Definition: The breakdown of glucose (6C) into two pyruvate molecules (3C) in the absence of oxygen.
• During energy investment requires 2 ATP to phosphorylate glucose.
• During cleavage, a 6 carbon sugar is split into two 3-carbon molecules
• During energy production, 4 ATP is produced (via substrate-level phosphorylation) 2 NADH is produced
• Glycolysis is the oxidation of Glyceraldehyde-3-phosphate to 3-phosphoglycerate.
• Step 6 reduces NAD* to NADH
• Step 7 produces ATP.
• Step 10 - the Final ATP-producing step (substrate-level phosphorylation).

Conversion and Location of Acetyl CoA

• Mitochondrial Matrix
• Reaction:Pyruvate+NAD++CoA→Acetyl CoA+NADH2
• Catalzyed by Pyruvate dehydrogenase complex. (enzyme with 60 subunits).

The Citric Acid Cycle

• Mitochondrial matrix that fully oxidizes Acetyl CoA to CO2, generating NADH & FADH2.
• Acetyl CoA (2C) combines with oxaloacetate (4C) to form citrate (6C).
• Two CO2 molecules are released per cycle.
• No ATP is directly produced, but 1 GTP is formed (converted to ATP).
• NADH & FADH2 carry high-energy electrons for oxidative phosphorylation.

Why the Citric Acid Cycle Depends on Oxygen

• The cycle itself does not use oxygen.
• However, oxygen is needed to regenerate NAD"+ and FAD.
• Without oxygen, NADH builds up thereby stopping the cycle.
• A pathway from oxygen occurs as the cycle proceeds in oxidative phosphorylation to recycle NAD +/FAD.

Comparing the Glycolysis and Citric Acid Cycle

Energy Balance of Glucose Oxidation

Citric Acid Cycle & Human Disease

• Mutations in SDH (Step 6) & FH (Step 7) cause:
  • Neurodegenerative diseases (encephalopathy, seizures)
  • Tumor formation (cancer risk)

Explore mRNA transport from the nucleus to ribosomes for protein synthesis. Learn about the roles of the 5' cap, poly-A tail, and nuclear pore complexes in ensuring the stability and export of mRNA. Understand how mRNA codons specify amino acids during translation.