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  1. Splicing Mutation
    • Mutations in RNA splice sites can cause inherited disease.
    • Mutations can eliminate splice donor or acceptor sites, or generate new sites.
    • Abnormal splicing then generates a defective protein.
    • ≈15% of single base-pair mutations that cause human disease result in mRNA splicing defects.
  2. RNA Export
    • All RNA processing occurs in the nucleus.
    • RNA is then transported from the nucleus to the cytoplasm for translation.
    • RNA is transported as a complex with proteins (ribonucleoprotein or RNP).
    • RNPs are actively transported through large channels in the nuclear envelope – nuclear pore
  3. Translation Roles
    • Bacterial translation is a target of common antibiotics,
    • Viruses like influenza co-opt translation for viral reproduction
    • Translational control in neurons may be especially important for learning and memory
  4. translation
    involves translating the genetic code specified by nucleotides (language of nucleic acids) into a linear chain of amino acids (language of proteins)
  5. genetic code
    • 61 codons aa, 3 STOP
    • 1. Nearly universal, exceptions (e.g. mitochondria) the same codons specify the same amino acids in all organisms. 2. It is degenerate (redundant). There are 61 triplets coding for only 20 amino acids, so more than one codon can specify the same amino acid.
    • 3. It is unambiguous. Each codon specifies only one amino acid.
    • 4. It is almost always read linearly and continuously. There are no breaks or overlaps of codons. The linear order of codons specifying the order of amino acids in a protein is referred to as the reading frame.
  6. Silent mutation
    • mutation from one codon to another that encodes the same amino acid.
    • For example, TTT to TTC mutation in DNA results in UUU to UUC codon change in mRNA but both encode phenylalanine so the protein would be the same.
  7. Missense mutation:
    • mutation that changes the codon so that it specifies a different amino acid
    • (GUA) instead of glutamate (GAA). This mutation causes sickle cell disease.
  8. Nonsense mutation
    • mutation that changes an amino acid-specifying codon to a STOP codon.
    • This type of mutation shortens the protein.
  9. Insertion or deletion, in-frame mutation
    • insertions or deletions of multiples of 3 nucleotides will result in addition or deletion of amino acids.
    • For example, the most common mutation that causes cystic fibrosis is a deletion of the codon specifying the phenylalanine at position 508.
  10. Frameshift
    insertion or deletion of nucleotides not divisible by 3 changes the reading frame downstream of the mutation
  11. tRNAs
    • Although distinct in sequence, all tRNAs have common structural characteristics:
    • 1. CCA sequence at the 3’ end where the amino acid attaches in a high energy bond.
    • 2. An anticodon loop containing a 3 nucleotide anticodon that base-pairs with the cognate codon in mRNA.
    • 3. Many bases other than A, C, U and G. 4. An L-shaped 3-dimensional structure formed by RNA double helices.
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  12. Aminoacyl tRNA synthetases
    • “charge” tRNAs with amino acids.
    • This is a 2 step process that requires ATP.
    • In the first step the tRNA synthetase catalyzes addition of ATP to the α-carboxy group of an amino acid to form the high energy intermediate aminoacyl-AMP.
    • The amino acid is then transferred to the 3’ or 2’ OH of the tRNA to generate aminoacyl-tRNA.
    • In this molecule the α-carboxy group of the amino acid is linked through a high energy ester bond to the 3’ or 2’ end of the tRNA.
    • Each tRNA synthetase is specific for one amino acid and one or a few tRNAs. Some tRNA synthetases have proof-reading mechanisms for removing incorrect amino acids from tRNA. Mistakes occur about once every 10^4 or 10^5 reactions.
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  13. Wobble
    • Charged tRNA recognizes the appropriate codon in mRNA because the anti-codon is complementary to the codon specifying the amino acid carried by that tRNA.
    • Anti-codon and codon interact through base-pairing.
    • Interaction follows the normal base-pairing rules (A:U, G:C) in the first 2 nucleotides of the codon but has “wobble” in the third position.
    • This allows degeneracy of the code so that a single tRNA can base-pair with multiple codons specifying an amino acid.
  14. Free ribosomes
    • translate proteins destined for the cytoplasm, nucleus, mitochondria, and peroxisomes.
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  15. Rough ER ribosomes
    translate proteins targeted for secretion, membranes of the secretory pathway, and lysosomes.
  16. Translation
    • proceeds in the 5’ to 3’ direction on mRNA
    • synthesizes proteins from the amino-terminus to the carboxy-terminus.
    • It can be divided into 3 stages: initiation, elongation, and termination.
  17. Initiation
    • involves association of tRNAi-Met, mRNA and the small (40S) subunit so that tRNAi-Met base-pairs with the Start codon. Then the 60S subunit associates to generate a translationally competent complex. Initiation is regulated by other proteins called initiation factors (eIF). tRNAi is a special tRNA for initiation – it is specific for methionine, is recognized by eIF and uniquely binds to the P site in the ribosome.
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  18. eIF2
    • GTPase switch protein.
    • It only associates with tRNA-Met and allows binding to the 40S subunit when bound to GTP.
  19. Cap binding factor (eIF4)
    binds to the 5’ cap of mRNA and brings the mRNA to the tRNAi-Met/40S complex, which then scans for AUG
  20. Formation of 80s Ribosome
    When tRNA(met) base-pairs with AUG then GTP is hydrolyzed and 60S large subunit associates.
  21. Elongation
    • involves entry of a new aa-tRNA, peptide bond formation, and translocation of the ribosome to the next codon. Involves elongation factors.
    • aa2-tRNA base-pairs with 2nd codon, regulated by GTPase switch
    • (Hydrolized when complementary codon match and moved to peptide bond formation)
    • Peptide bond formation catalyzed by 28S RNA.
    • Ribosome translocates along mRNA to bring 3rd codon into place for next aa-tRNA
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  22. Termination
    • involves recognition of the STOP codon, cleavage of the polypeptide from the tRNA, and dissociation of the mRNA and ribosomal subunits.
    • Release factors bind to STOP codon.
    • Peptide is released by peptidyl transferase activity of 28S rRNA.
    • Dissociation of tRNA, mRNA and ribosomal subunits
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  23. Polysomes
    Multiple individual ribosomes can translate mRNA at the same time, increasing the efficiency of translation.
  24. Gentamycin
    Some individuals carry a mutation in the mitochondrial small subunit RNA (12S) that make them particularly sensitive to gentamycin. These individuals can suffer from antibiotic induced deafness.
  25. RNA interference
    • A new mode of gene regulation has been recently discovered that involves small doublestranded RNA.
    • double-stranded RNA interferes with gene expression
    • microRNA involved in gene silencing.
    • MicroRNAs are important during development to silence particular geneand allow proper differentiation of specific cetypes.
    • Exogenous double-stranded RNA can also be introduced into cells to silence a targeted gene.
    • This allows an easy method to turn off expression of a specific gene.
    • Such “targeted” RNAi has therapeutic potential to treat diseases such as cancer and infection by RNA viruses such as HIV.
  26. components of RNAi
    • the RNA,
    • two processing RNases, one in the nucleus and one in the cytoplasm, cleave the precursor to produce the short (21-25 nucleotide) double-stranded RNA
    • and the RNA-induced Silencing Complex (RISC) that actually carries out silencing
    • Endogenous microRNA is synthesized as part of a longer RNA by RNA pol II. The microRNA sequences are homologous and base-pair, forming a “stem” in the precursor.
  27. RISC
    • This RNA is unwound and one strand associates with an Argonaut protein, which is part
    • The single-stranded microRNA targets RISC to mRNA that contain homologous sequences by base-pairing. Protein production from the mRNA is prevented either by mRNA degradation or by RISC-mediated inhibition of mRNA translation.
  28. secretory pathway
    • consists of the endoplasmic reticulum (ER) and Golgi complex
    • responsible for transport of newly-synthesized proteins to the appropriate compartment
    • excretion
    • receptors
    • organelles
    • 1) protein synthesis and translocation across (or into) ER membrane.
    • 2) Protein folding and modification in ER
    • 3) Transport to Golgi, lysosomes, plasma membrane by budding and fusion of small transport vesicles.
  29. endocytic pathway
    • consists of endosomes (early and late) and ends at the lysosome.
    • involved in uptake of material from the plasma membrane and the extracellular fluid.
    • It is important for uptake of nutrients such as cholesterol and iron, as well as internalization of signaling receptors.
    • This pathway is also used by certain pathogens such as viruses to enter the cell
  30. Smooth ER
    major site of fatty acid and lipid synthesis
  31. Rough ER
    studded with ribosomes, site of synthesis of proteins that enter the secretory pw (also some lipid synthesis) and protein glycosylation.
  32. Golgi complex
    • complex organelle with both tubulated regions and stacked membrane disks (called cisternae).
    • Organized into cis Golgi network (closest to ER), cis, medial and trans Golgi cisternae, and trans Golgi network (closest to PM).
    • Site of protein glycosylation and modification of carbohydrate side chains.
    • Also major site of protein sorting.
  33. Sorting Signal
    • Targeting relies on a short stretch of amino acids in the nascent polypeptide that directs it to the appropriate compartment.
    • Referred to as a sorting signal because it is responsible for sorting to a specific compartment.
  34. signal sequence
    • Sorting signal for ER targeting
    • normally located at the N-terminus of the protein and consists of one or more positivelycharged amino acids followed by 6-12 hydrophobic amino acids
    • Signal sequences bind to proteins that are associated with the ER
  35. Signal Recognition Particle
    • SRP is ribonucleoprotein particle with one RNA and multiple polypeptides.
    • Binds to the large ribosomal subunit and the signal sequence as emerges from ribosome, pauses translation
    • SRP-ribosome-nascent polypeptide complex is then bound by SRP receptor, an ER membrane protein
    • Both SRP and SRP receptor are GTPases – GTP binding by both increases affinity of SRP for SR.
    • Once bound to SRP receptor, the ribosome and nascent chain are transferred to the translocon, a protein that forms a gated channel through the ER membrane.
    • SRP and SRP receptor hydrolyze GTP and dissociate.
    • Nascent polypeptides enter the ER through a protein channel.
    • Newly synthesized membrane proteins integrate into the ER from the translocon
    • Same basic process but a hydrophobic sequence within the protein is transferred to the membrane from the translocon, resulting in an integral membrane protein
  36. N-linked glycosylation in the ER
    • Proteins undergo N-linked glycosylation during translocation and fold, including formation of disulfide bonds, in the lumen of the ER.
    • Glycosylation involves the addition of a preformed 14 sugar oligosaccharide from a lipid-linked donor that is embedded in the ER membrane through the lipid (dolichol).
    • The sugar is transferred in block as the target amino acid sequence (asparagine –X – serine/threonine) emerges from the translocon.
    • This “core” oligosaccharide is conserved in all eukaryotic species.
    • Core is trimmed by glycosidases in the ER
  37. Vesicle formation
    • Coat formation: polymerization of specific proteins onto the cytoplasmic face of the organelle membrane forms a “coat” that drives membrane curvature and collects proteins (cargo) for transport to the next compartment.
    • Sorting signals (normally 4-6aa in length) on the cytoplasmic domains of cargo bind to coat proteins, directing the cargo into the forming vesicle. Once the vesicle buds from the donor compartment, the coat is released (uncoating).
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  38. 3 types of coats
    • COPI (Golgi to ER, intra-Golgi)
    • COPII, (ER to Golgi)
    • clathrin (Golgi to endosomes; endocytosis)
  39. Vesicle docking and fusion
    • Vesicles are targeted to the correct recipient organelle membrane by two classes of proteins:
    • Tethers - which provide the initial link between vesicle and target
    • SNAREs – which are integral membrane proteins present on both the vesicle (v-SNARE) and target membrane (t-SNARE) and bind each other to drive membrane fusion.
    • v-SNAREs and t-SNAREs form parallel α-helical bundles that force the vesicle and target membrane together. Different sets of tethers and SNAREs used at each transport step, providing specificity in docking and fusion.
  40. modifications of N-linked oligosaccharides
    • Golgi complex carries out. After proteins are transported from the ER to the Golgi, N-linked glycoprotein oligosaccharides are modified by glycosidases (cleave sugars) and glycosylases (add sugars).
    • Enzymes are located in distinct Golgi cisternae so that modification progresses as cargo moves through the cisternae from cis to trans.
  41. trans Golgi network (TGN)
    • major site of protein sorting
    • Secreted proteins sorted to constitutive secretory vesicles or, in specialized cells to regulated secretory vesicles or granules (e.g. insulin, glucagon, neurotransmitters, digestive enzymes).
    • Regulated secretory vesicles only fuse to plasma membrane if cell receives the appropriate extracellular signal. Lysosomal enzymes sorted into clathrin coated vesicles that fuse with endosome
  42. Clathrin coated vesicles
    • mediate sorting of lysosomal enzymes from the TGN to endosomes.
    • Clathrin 3D structure is designed to assemble into polyhedral coats on a membrane.
    • Clathrin coats also contain “adaptor complexes” which link clathrin to membrane and bind cargo.
    • COPI and COPII coats have similar 2-tiered structure.
  43. Lysosomal Sorting Signal
    • A sugar modification on N-linked oligosaccharides is the sorting signal on lysosomal enzymes.
    • Oligosaccharide side chains on lysosomal enzymes are modified by addition of phosphate to the carbon 6 of mannose (mannose-6-phosphate or M6P) in a two step reaction catalyzed by protein N-acetylglucosamine phosphotransferase (GlcNAC transferase).
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  44. Sorting of lysosomal enzymes with M6P.
    • 1. M6P is recognized by a transmembrane M6P receptor. Receptor is collected by clathrin coats at the TGN through interactions between amino acid sorting signal and clathrin adaptor. Clathrin coated vesicle buds.
    • 2. Unocoating
    • 3. Uncoated vesicle docks and fuses with late endosome membrane.
    • Low pH of endosome (≈pH 5) causes enzyme to be released from receptor.
    • 4. Enzyme is delivered to lysosome.
    • 5. Receptor is returned to TGN by vesicles carrying a different type of coat.
    • Defects in lysosomal enzyme sorting due to mutation of GlcNAC phosphotransferase cause recessive inherited lysosomal storage disease called I-cell disease.
    • I stands for “inclusions” that are observed by microscopy – the lack of lysosomal enzymes leads to large accumulation of undegraded material in lysosomes that forms the inclusions.
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