Biochemistry - molecular

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Biochemistry - molecular
2013-03-22 17:31:02

Biochemistry (molecular)
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  1. DNA organization
    DNA → Nucleosomes → Chromatin → Chromosomes
    • Negatively charged DNA loops twice around positively charged histone octamer to form nucleosome
    • Histones: rich in lysine (+), arginine (+)
    • H1 is only histone not in nucleosome core
  2. Heterochromatin
    • Condensed, transcriptionally inactive, sterically inaccessible
    • **HeteroChromatin = Highly Condensed
  3. Euchromatin
    • Less condensed, transcriptionally active, sterically accessible
    • Eu = true, "truly transcribed"
  4. DNA methylation
    Templante strand cytosine and adenine are methylated in DNA replication, which allows mismatch repair enzymes to distinguish between old and new strands in prokaryotes
  5. Histone methylation
    • Inactivates transcritpion of DNA
    • **Methylation makes DNA Mute
  6. Histone acetylation
    • Relaxes DNA coiling, allowing for transcription
    • **Acetylation makes DNA Active
  7. Nucleotides
    • PURines (A, G) - 2 rings
    • PYrimidines (C, T, U) - 1 ring
    • **PURAGold; CUT the PY (pie)

    • -Deamination of cytosine → uracil
    • -Uracil: found in RNA
    • -Thymine: found in DNA

    • G☰C; stronger bond than A=T bonds
    • -↑G-C content means ↑ melting temperature
    • Amino acids necessary for purine synthesis:
    • **GAG
    • Glycine
    • Aspartate
    • Glutamine
  8. Pyrimidine
  9. NucleoSide
    NucleoSide = base + ribose (Sugar)
  10. NucleoTide
    • NucleoTide = base + ribose + phosphaTe
    • Linked by 3'-5' phophodiesterase bond
  11. De novo purine synthesis
    • Start with sugar + phosphate (PRPP)
    • PRPP synthetase (inhibited by 6-MP)
    • Add base
  12. De novo pyrimidine synthesis
    • Make temporary base (orotic acid)
    • Add sugar + phosphate (PRPP)
    • Modify base
    • Rate limiting step: ATP + CO2 + glutamine → carbamoyl phosphate (enzyme: CPSII)
  13. CPSII
    • Found in cytosol
    • Involved with pyrimidine synthesis
    • Nitrogen from flutamine
  14. Various anti-neoplastic antibiotic drugs interfere with purine synthesis
    • Hydroxyurea inhibits ribonucleotide reductase
    • 6-mercaptopurine (6-MP) blocks de novo purine synthesis
    • 5-fluorouracil (5-FU) inhibits thymidylate synthase (↓ deoxythymidine monophophate [dTMP])
    • Trimethoprim (TMP) inhibits bacterial dihydrofolate reductase (↓ dTMP)
  15. Orotic aciduria
    cause, finding, treatment
    • Inability to convert orotic acid to UMP (de novo pyrimidine synthesis pathway)
    • -defect in UMP synthase; Autosomal recessive

    • Findings:
    • -↑ Orotic acid in urine

    • -Megaloblastic anemia (does NOT improve with administration of Vit B12 or folic acid)
    • -Failure to thrive
    • -No hyperammonemia (vs. OCT deficiency [urea cycle enzyme deficiency]: ↑ orotic acid with hyperammonemia)

    • Tx:
    • -Oral uridine administration
  16. Ribonucleotide reductase
    • Enzyme used to convert ribonucleotides to deoxyribonucleotide
    • inhibited by hydroxyurea
  17. Rate-limiting step in purine synthesis?
    Glutamine PRPP Aminotransferase
  18. Rate limiting step in pyrimidine synthesis
  19. What are the sources of carbons in the formation of purines?
    • CO2
    • Glycine
    • Tetrahydrofolate
  20. What are the carbon sources in pyrimidine synthesis?
    • Aspartate
    • CO2
  21. What accounts for the positive charge of histones? What accounts for the negative charge of DNA?
    • Histones: lysine and arginine
    • DNA: phophate groups
  22. What strand of DNA nucleotides opposes this DNA strand: 5'-ATTGCGTA-3'?
    • 5'-TACGCAAT-3'
    • ALWAYS write 5'-3'
  23. Which medication matches the following statement?
    • Inhibits ribonucleotide reductase: Hydroxyurea
    • Inhibits dihydrofolate reductase: TMP, MTX 
    • Inhibitds thymidylate synthase: 5-FU
    • Inhibits inosine monophophate dehydrogenase: micophinalate
    • Inhibits PRPP synthetase: 6-MP
  24. What are the characteristic features of orotic aciduria?
    • Orotic acid in urine
    • Megaloblastic anemia, not correctable by B12 or Folate
    • Failure to thrive
    • No elevation of ammonia level
  25. Purine salvage
    process of breaking down purines
    • End product: uric acid
  26. Xanthine oxidase
    What drug inhibits this?
    • Allopurinol inhibits xanthine oxidase
    • Cancer drug that is metabolized by xanthine oxidase: 6-MP
    • Immunosuppresant that is metabolized by xanthine oxidase: Azothioprine

    **administering allopurinol with 6-MP/azothioprine makes the 6-MP/azothiprine more toxic
  27. Lesch-Nyan syndrome
    purine salvage deficiency
    • Defective purine salvage owing to absent HGPRT, which converts hypoxanthine to IMP and guanine to GMP
    • **He's Got Purine Recovery Trouble
    • -Hypoxanthine-guanine phosphoribosyltransferase

    • Findings: excess uric acid production and de novo purine synthesis
    • -X-linked recessive
    • -Retardation
    • -Self-mutilation (lip biting)
    • -aggression
    • -hyperuricemia
    • -gout
    • -choreoathetosis

    • Tx: allopurinol
    • -gets rid of excess uric acid
  28. Adenosine deaminase deficiency
    • Excess ATP and dATP imbalance nucleotide pool via feedback inhibition of ribonucleotide reductanse
    • Results: prevents DNA synthesis → ↓ lymphocyte count
    • Major cause of SCID
    • Autoimmune recessive - 1st disease to be treated by experimental human gene therapy
  29. SCID
    • Severe Combined Immunodificiency Disease
    • Happens to kids - 7 different genetic causes
    • Features:
    • -Severe recurrent infections
    • -Chronic diarrhea
    • -Failure to thrive
    • -Absence of thymic shadow on CXR
  30. Genetic code features
    • Unambigous: each codon speficies only 1 amino acid
    • Degenerate/redundant: most amino acids are coded by multiple codons
    • ---Exceptions: methionine and tryptophane (AUG and UGC, respectively)
    • Commaless, nonoverlapping: read from fixed starting point as continuous sequence of bases
    • ---Exception: some viruses
    • Universal: Genetic code is conserved throughout evolution
    • ---Exception: human mitochondria
  31. Point mutations in DNA
    Severity of damage
    • Silent: same amino acid, often base change in 3rd position ("tRNA wobble")
    • Missense: changed amino acid (conservative - new amino acid is similar in chemical structure)
    • Nonsense: Change results in early stop codon **Stop the nonsense
    • Frameshift: Change results in misreading of all nucleotides dowstream, usually resulting in a truncated, nonfunctional protein
    • Severity of damage: silent < missense < nonsense < frameshift
  32. How does UV light damage DNA?
    UV light pairs thymine; creates thymine dimers on the same strand of DNA
  33. DNA replication
    • Eukaryotic DNA replication is complex (more so than prokaryotic DNA replication)
    • Semiconservative, involves both continuous and discontinuous (Okazaki fragment) synthesis
  34. Origin of replication
    • Particular consensus sequence of base pairs in genome where DNA replication begins
    • May be single (prokaryotes) or multiple (eukaryote)
  35. Replication fork
    Y-shaped region along DNA template where leading and lagging strand are synthesized
  36. Helicase
    Unwinds DNA template from replication fork
  37. Single-stranded binding proteins
    • Prevent strands from reannealing
    • stabilize the unbound DNA
  38. DNA topoisomerases
    • Create a nick in the helix to relieve supercoils created during replication
    • **Fluoroquinolones: inhibit DNA gyrase (prokaryotic topoisomerase II)
    • **Etoposide: inhibits eukaryotic topoisomerase (anti-neoplastic)
    • **Anti-Scl-70: antibody to topoisomerase; associated with diffuse scleraderma
  39. Primase
    Makes an RNA primer on which DNA polymerase III can initiate replication
  40. Eukaryote DNA polymerase
    • α-replicates laggging strand, synthesis of RNA primer
    • β-repairs DNA
    • γ-replicates mitochondrial DNA
    • δ-replicates leading strand DNA
    • ε-repairs DNA
  41. DNA polymerase III
    • Prokaryotic only
    • Elongates leading strand by adding deoxynucleotides to the 3' end
    • Elongates lagginst strand until it reaches primer of preceding fragment
    • 3'→5' exonuclease activity "proofreads" each added nucleotide
    • **DNA polymerase III has 5'→3' synthesis and proofreads with 3'→5' exonuclease
  42. DNA polymerase I
    • Prokaryotic only
    • Degrades RNA primer; replaces it with DNA
    • **Same function as DNA polymerase III, but also excises RNA primer with 5'→3' exonuclease
  43. DNA ligase
    • Catalyzes the formation of phosphodiesterase bond within a strand of double-stranded DNA
    • Joins Okazaki fragments
    • Seals
  44. Telomerase
    Enzyme adds DNA to 3' end of chromosomes to avoid loss of genetic material with every duplication
    • DNA replication
  45. DNA repair
    single vs double strand
    • Single strand:
    • -Nucleotide excision repair
    • -Base excision repair
    • -Mismatch repair

    • Double strand:
    • -Nonhomologous end joining
  46. Nucleotide excision repair
    • Specific endonucleases release the oligonucleotide-containing damaged bases
    • DNA polymerase and ligase fill and reseal the gap, respectively
    • Repairs bulky helix-distorting lesions
    • *Mutated in xeroderma pigmentosumwhich prevents repair of pyrimidine dimers because of UV light exposure
  47. Base excision repair
    • Specific glycosylases recognize and remove damaged bases, apurinic/apyrimidinec endonucleases cuts DNA at both apurinic and apyridiminic sites
    • Empty sugar is removed
    • gap is filled and resealed
    • Important in repair of spontaneous/toxic deamination
  48. Mismatch repair
    • Newly synthesized strand is recognized
    • mismatched nucleotides are removed
    • gap is filled and resealed
    • **Mutated in Hereditary nonpolyposis colorectal cancer (HNPCC) lynch syndrome
  49. Nonhomologous end joining
    • Brings together 2 ends of DNA fragment to repair double-stranded breaks
    • No requirement for homology
    • **Mutated in ataxia telangiectasia
  50. DNA/RNA/protein synthesis direction
    • DNA and RNA are synthesized 5' → 3'
    • 5' end bears the triphophate (energy source for bond)
    • Triphosphate bond is the target of the 3' hydroxyl attack
    • Drugs blocking DNA replication often have modified 3'OH, preventing addition of the next nucleotide ("chain termination")
    • mRNA is read 5' → 3'
    • protein synthesis: N-terminus to C-terminus
  51. Types of RNA
    • rRNA is the most abundant type (synthesized in the nucleolus)
    • mRNA is the longest type (synthesized in nucleoplasm)
    • tRNA is the smallest type  (synthesized in nucleoplasm)
    • **rampant, massive, tiny
  52. start and stop codons
    • mRNA start codons: AUG (or rarely GUG)
    • **AUG inAUGurates protein synthesis
    • -Eukaryotes → methionine
    • -Prokaryotes → formylmethionine (f-met)

    • mRNA stop codons: UGA, UAA, UAG
    • -U Go Away
    • -U Are Away
    • -U Are Gone
  53. Functional organization of the gene
  54. Regulation of gene expression
    promoter, enhancer, silencer
    • Promoter:
    • -Site where RNA polymerase and multiple other transcription factors bind to DNA upstream from gene locus
    • -AT-rich sequence with TATA and CAAT boxes
    • *Mutations: dramatic ↓ in amount of gene transcribed

    • Enhancer:
    • -Stretch of DNA that alters gene expression by binding transcription factors

    • Silencer:
    • -Site where negative regulators (repressors) bind

    Enhancers and silencers may be close to, far from, or even within (in an intron) the gene whose expression it regulates
  55. Operon
    • Structural genes that are transcribed + promoter region + all regulatory regions
    • i.e. Lac operon
  56. RNA polymerases
    • RNA polymerase I makes rRNA
    • RNA polymerase II makes mRNA; opens DNA at promotor site
    • -α-amanitin, found in Amanita phalloides (death cap mushrooms) inhibits RNA polymerase II; severe hepatotoxicity if ingested
    • RNA polymerase III makes tRNA
    • *No proofreading function
    • Can initiate chains
    • **I, II, and III are numbered as their products are used in protein synthesis
  57. RNA polymerase
    • 1 RNA polymerase (multisubunit complex)
    • makes all 3 kinds of RNA
    • **Rifampin: antibiotic that inhibits prokaryotic RNA polymerase
  58. RNA processing

    • hterogenous nuclear RNA (hnRNA): initial transcription
    • -hnRNA destined for translation is called pre-mRNA

    • Processing occurs in nucleus; only processed RNA is transported out of the nucleus
    • After transcription:
    • -Capping on 5' end (addition of 7-methylguanosine cap)
    • -Polyadenylation on 3' end (~200A's); Poly-A polymerase does not require a template (AAUAAA = polyadenylation signal)
    • -Splicing out of introns
  59. Splicing of pre-mRNA
    • 1. Primary transcript combines with smRNPs and other proteins to form splicosome
    • 2. Lariate-shaped (looped) intermediate is generated
    • 3. Lariate is released to remove intron precisely and join 2 exons
    • **Pts with SLE (lupus) make antibodies to spliceosomal snRNPs
  60. Splicing of pre-mRNA
  61. Introns vs Exons
    • Exons: actual genetic information coding for protein (exons exit the nucleus and are expressed)
    • Introns: intervening noncoding segments of DNA (introns are intervening sequences and stay in the nucleus)
    • Different exons can be combined by altering splicing to make unique proteins in different tissues (e.g. β-thalassemia mutations)
  62. tRNA
    • 75-90 nucleotides, 2° structure, cloverleaf form, anticodon end is opposite 3' aminoacyl end
    • All tRNAs have CCA at 3' end along with a high percentage of chemically modified bases
    • Amino acid is covalently bound to the 3' end of the tRNA
    • *CCA: Can Carry Amino acids
  63. tRNA
    • Aminoacyl-tRNA synthetase (1 per amino acid, "matchmaker", uses ATP) scrutinizes aa before and after it binds to tRNA
    • Incorrect: bond is hydrolyzed
    • The aa-tRNA bond has energy for formation of peptide bond
    • A mischarged tRNA reads usual codon but inserts wrong amino acid
    • *Aminoacyl-tRNA synthetase and binding of charged tRNA to the codon are responsible for accuracy of amino acid selection
    • **Tetracyclines bind 30S subunit, preventing attachment of aminoacyl-tRNA
  64. tRNA wobble
    • Accurate base pairing is required only in the first nucleotide positions of an mRNA codon
    • codons differing in the 3rd "wobble" position may code for same tRNA/amino acid (as a result of degeneracy of genetic code)
  65. Eukaryotic ribosome
  66. Protein synthesis
    Activated by GTP hydrolysis, initiation factors (IFs) help assemble the 40S ribosomal subunit with the initiator tRNA and are released when the mRNA and the ribosomal subunit assembles with the complex

    • *ATP-tRNA Activation (charging)
    • GTP-tRNA Gripping and Going places (translocation)
  67. Ribosomal subunits
    Eukaryotes vs prokaryotes
    • Eukaryotes: 40S + 60S → 80S (Even)
    • PrOkaryotes: 30S + 50S → 70S (Odd)
  68. Protein synthesis
    • 1. Aminoacyl-tRNA binds to A site (except for initiator methionine)
    • 2. Ribosomal rRNA ("ribozyme") catalyzes peptide bond formation, transfers growing polypeptide to amino acid in A site
    • 3. Ribosome advances 3 nucleotides toward 3' end of mRNA, moving peptidyl tRNA to P site (translocation)

    • **Think of "going APE"
    • -A site = incoming Aminoacyl-tRNA
    • -P site = accommodates growing Peptide
    • -site = holds Empty tRNA as it Exits
  69. Protein synthesis
    • Stop codon is recognized by release factor
    • completed protein is released from ribosome
  70. Antibiotics that act as protein synthesis inhibitors
    • Aminoglycosides: bind 30S and inhibit formation of initiation complex and cause misreading of mRNA
    • Tetracyclines: bind 30S and block aminoacyl tRNA from entering the acceptor site
    • Chloramphenicol: binds 50S and inhibits peptidyl transferase
    • Macrolides: bind 50S and prevent release of uncharged tRNA after it has donated its amino acid
  71. Posttranslational modifications
    • Trimming: Removal of N- or C-terminal propeptides from zymogens to generate mature proteins
    • Covalent alterations: Phosphorylation, glycosylation, hydroxylation, methylation, acetylation
    • Proteasomal degradation: Attachment of ubiquitin to defective proteins to tag them for breakdown
  72. DNA repair defects
    • Xeroderma pigmentosum: hypersensitivity to UV light → 1000x increase risk for skin cancer
    • Ataxia-telangiectasia: sensitivity to ionizing radiation, immunodeficiency, ataxia beginning at 1-2 years
    • Bloom's syndrome: hypersensitivity to sunlight, leukemias and lymphomas are common, average age of cancer onset 25
    • Hereditary Nonpolyposis Colorectal Cancer (HNPCC): lynch syndrome
    • BRCA1 and BRCA2: breast, uterine cancer risk
  73. which antibiotic matches the following description?
    • Inhibits 50S peptidyltransferase: Chloramphenicol, streptogramins
    • Binds 50S, blocking translocation: Macrolides, Linezolid
    • Bind 30S, preventing attachment of tRNA: Tetracyclines
    • Inhibits prokaryotic RNA polymerase: Rifampin
    • Inhibits prokaryotic topoisomerase: Fluoroquinolones
    • Inhibits prokaryotic dihydrofolate reductase: TMP
  74. 3 different mechanisms cells employ to breakdown proteins?
    • Ubiquitin mechanism
    • Lysosomal degradation mechanism
    • Calcium-dependent enzyme
  75. What enzyme catalyzes peptide bond formation during protein synthesis?
    Peptidyltransferase (type of ribosime)
  76. What enzyme matches amino acids to tRNA?
    Aminoacyl tRNA synthetase
  77. What are the mRNA stop codons?
    • UGA
    • UAA
    • UAG
  78. What are the different RNA polymerases in euchariotes?
    • RNA polymerase I: codes rRNA
    • RNA polymerase II: transcribes mRNA
    • RNA polymerase III: transcribes tRNA
  79. What amino acid frequently has more coding sequences in the mRNA than are represented in the peptide that is created from that mRNA?
    • Methionine
    • more AUG than methionine in the protein, commonly cleaved off
  80. How is hnRNA processed before it leaves the nucleus?
    • 5' cap
    • Poly-A tail
    • Splicing out of the intron
  81. What are the characteristic sequences of the promotor region? What does a mutation in the sequencec cause?
    • -25 TATA box
    • -75 CAT box

    mutations cause decrease in amount of gene that is transcribed
  82. What enzyme is deficient in Lesch-Nyhan syndrome? What is the treatment?
    • HGPRT;
    • Tx: allopurinol
  83. What structural motifs allow for proteins to bind to DNA?
    • Helix turn helix
    • helix loop helix
    • leucine zippers
    • zinc fingers