Exam 3 Flashcards

Translation Overview

• Translation is the process by which messenger RNA (mRNA) is translated into a polypeptide chain (protein).

• Occurs in the cytosol where mRNA base pair sequences are translated into amino acid sequences.

• Nucleotides are read in groups of three known as codons.

• There are 64 codons made from 4 different bases (A, U, C, G) with each codon specifying either a start, stop signal, or an amino acid.

• The relationship between a codon and its corresponding amino acid is established by the genetic code.

tRNA Molecule

• tRNA (transfer RNA) molecules serve as the essential link between codons on mRNA and their corresponding amino acids.

• Each tRNA is approximately 80 nucleotides in size, featuring an anti-codon region that binds to its complementary codon on mRNA.

Synthesis and Modifications of tRNA

• tRNA molecules are synthesized by RNA polymerase III (POL III) as large transcripts that are subsequently trimmed at both ends and potentially modified in the middle.

• These modifications create non-traditional bases (Ψ, Im, Gm, I, T), which contribute to the unique final conformation of the tRNA.

Mechanism of tRNA and Amino Acid Binding

• Aminoacyl Synthetase is an enzyme that catalyzes the linking of an amino acid to its specific tRNA, forming a high-energy bond.

• The amino acid-activated tRNA is crucial for peptide bond formation during translation.

• The reaction consumes energy from ATP, producing AMP and two inorganic phosphates (Pi).

Hydrolytic Editing

• Prior to catalyzing peptide bond formation, tRNA synthetases can proofread and edit tRNAs, removing incorrect amino acids to maintain fidelity in protein synthesis.

Ribosome Structure and Function

• The ribosome is a large ribonucleoprotein complex composed of two subunits (large and small) made up of several rRNAs and over 50 proteins.

• Eukaryotic ribosomes assemble in the nucleolus and are exported into cytosol for protein synthesis. Millions of ribosomes exist per eukaryotic cell.

• The small subunit contains an mRNA binding site and multiple binding sites for tRNAs, while the large subunit catalyzes peptide bond formation.

Eukaryotic Initiation of Translation

• The initiation process starts with the initiator aminoacyl-tRNA binding to the small ribosomal subunit.

• Additional initiation factors (eIFs) bind to guide this small ribosomal subunit to the 5’ cap of mRNA.

• The small subunit scans the mRNA for the AUG start codon, driving movement powered by ATP hydrolysis.

Leaky Scanning in Eukaryotes

• When AUG is encountered, initiation factors dissociate, and the large ribosomal subunit binds, starting translation.

• Sometimes, the first one or two AUGs can be ignored in favor of a later AUG, a phenomenon called leaky scanning, which allows for the synthesis of functionally identical proteins with different localization signals.

Bacterial mRNA and Translation

• Bacterial mRNAs lack a 5’ cap and instead utilize consensus sequences for small subunit ribosome attachment, which leads to polycistronic mRNAs.

• This allows for the translation of multiple proteins from a single mRNA strand.

Elongation of the Polypeptide Chain

• Elongation continues with incoming aminoacyl-tRNAs bringing new amino acids to the A-site of the ribosome.

• Energy from the breaking of high-energy bonds assists in peptide bond formation, while the large ribosomal subunit catalyzes the reaction, shifting the ribosome down the mRNA template without moving the tRNAs.

Translation Termination

• Stop codons (UAA, UAG, UGA) signal termination of translation, binding release factors (RFs) that mimic tRNAs.

• These RFs introduce a water molecule instead of an amino acid, triggering the release of the newly synthesized polypeptide and terminating translation.

Polyribosomes (Polysomes) and Efficiency

• Polyribosomes are formations of multiple ribosomes translating the same mRNA simultaneously, enhancing the rate of protein synthesis in both prokaryotes and eukaryotes.

• In eukaryotes, polyribosomes can be formed after transcription is completed, but bacteria begin translation even before transcription ends.

Protein Folding and Quality Control

• Protein folding begins during synthesis, with the assistance of molecular chaperones (hsp60 and hsp70) that help proteins reach their correct conformations.

• Proteins that cannot be properly folded are marked for degradation by proteasomes and ubiquitin chains, ensuring cellular quality control by removing defective proteins.

Membrane Structure

Cell Membranes

• Plasma membrane (in prokaryotes and eukaryotes)

  • Defines cellular space and boundaries.

  • Maintains biochemical and electrical differences.

  • Acts as a conduit for extracellular communication.

• Organelle membranes (found in eukaryotes only):

  • Define organelle lumens and boundaries.

• Membrane composition:

  • Lipid bilayer

  • Many inserted and surface-associated proteins

  • A few other types of molecules

Lipid Bilayer

• Membrane lipids are amphipathic molecules:

  • Contain both polar and non-polar bonds.

• Membrane lipids have a unique shape:

  • Hydrophilic “head” region (polar) and

  • Hydrophobic “tail” region (non-polar).

• Membrane lipids spontaneously form bilayers in aqueous solution.

Spontaneous Bilayer Formation

• Energetically Unfavorable: Lipid micelle (edges exposed to water).

• Energetically Favorable: Sealed compartment formed by phospholipid bilayer.

Phospholipids

• Most abundant lipid in cell membranes.

• Many types of phospholipids:

  • Four major types found in cell membranes.

Four Major Phospholipids

• Phosphatidyl-ethanolamine

• Phosphatidyl-serine

• Phosphatidyl-choline

• Sphingomyelin

Lipid Bilayer is Fluid

• Individual lipids diffuse laterally.

• Rotate rapidly about their axis.

• Nonpolar tails are extremely flexible.

Cells Regulate Bilayer Fluidity

• Through:

  • Cis-double bonds in fatty acid tails.

  • Addition of other types of molecules.

• Allows cells to maintain constant fluidity independent of temperature fluctuations.

Lipid Bilayer Fluidity

• Affected by the addition of certain non-phospholipid amphipathic molecules, e.g., cholesterol:

  • Interacts with phospholipids to increase rigidity between tails near head region.

  • Lower ends of tails remain flexible and disrupt attractive interactions between phospholipids.

  • This prevents crystallization (freezing).

Lipid Rafts

• Weak associations between lipids, often include cholesterol and proteins.

  • Lipid rafts drift as a group.

  • Membrane in raft region is often thicker, may serve as a mechanism for holding membrane proteins.

Glycolipids

• Lipid molecules + attached sugar molecules.

• Found only on non-cytoplasmic surface (extracellular and intra-organelle).

Glycolipid Functions

• Protection.

• Charged glycolipids concentrate ions at cell surface.

• Cell recognition.

• Unintended function: may allow bacterial toxins to enter cells.

Membrane Proteins

• Define much of membrane function.

• Amount and type of protein in any region of the membrane is highly variable.

• Membrane proteins often have oligosaccharide chains attached to their non-cytosolic domains (similar to glycolipids).

Mechanisms of Association

• Transmembrane proteins:

  • Always amphipathic; may be single or multi-pass.

  • Spanning segment often α-helix or β-barrel.

• Surface proteins:

  • Usually located in cytosolic region; sometimes extracellular.

  • Variety of attachment mechanisms include:

    • α-helix associated with lipid bilayer.

    • Covalent attachment via oligosaccharide chain.

    • Noncovalent attachment to transmembrane protein.

Peripheral vs Integral Proteins

• Peripheral proteins: Weak noncovalent surface attachment.

• Integral proteins:

  • Transmembrane or covalently attached to the surface.

  • All peripheral proteins are surface proteins, but not all surface proteins are peripheral.

  • All transmembrane proteins are integral but not all integral proteins are transmembrane.

Examples of Membrane Proteins

• Spectrin:

  • Cytosolic and noncovalently associated (peripheral) protein found in red blood cells.

  • Associates with cytoskeleton and other proteins to form a flexible mesh, maintaining cell shape (concave red blood cell).

• Glycophorin:

  • Transmembrane red blood cell protein with a membrane-spanning α helix domain.

  • Extensive extracellular domain composed of oligosaccharide chains, providing adaptability for motility.

• Bacteriorhodopsin:

  • Multi-pass transmembrane protein functioning as a proton pump in archaea.

  • Contains retinal, responding to photons and enabling mechanical work to pump protons out of the cell.

Membrane Protein Complexes

• Include several protein subunits such as cytochrome, forming complex cellular structures that participate in various membrane functions.

Glycocalyx

• Extracellular surface covered with sugars.

• Covalently attached to:

  • Membrane proteins (glycoproteins)

  • Lipids (glycolipids)

• Functions:

  • Provides protection against mechanical damage

  • Facilitates cell recognition (as further discussed in Chapter 19).

Proteoglycan

• A type of glycoprotein in which one or more attached sugars are amino sugars (e.g., glycosaminoglycan).