Biochem Exam #1

  1. Griffith
    • -Genetic components are not killed by heat
    • -Transformation and recombination can occur
    • -Proved genetic component existed
  2. Avery, MacLeod, McCarty
    -DNA is destroyed by DNAse not RNAse or proteases
  3. Nucleosome
    H2A/H2B/H3/H4 x 2

  4. Solenoid
    -Nucleosome + Linker DNA + H1 Histones
  5. Chromatin
    -DNA + associated nucleoproteins
  6. Histones
    -Small basic proteins (Lys, Arg), + charged
  7. Junk DNA
    • -Regulates transcription/translation of protein coding sequences
    • -Highly conserved
    • -May be involved: chromosome structure/centromere function/homolog recognition
    • -Evolution
    • -Vital role for organisms to adapt to changes
  8. mRNA, tRNA, rRNA %
    • -mRNA 5%
    • -tRNA 10%
    • -rRNA 85%
  9. Small RNA
    • -Nucleus: splices hnRNA
    • -Cytosol: targets proteins
  10. How is the double helix stabilized?
    • -Hydrogen bonding
    • -Hydrophobic stacking
  11. 2nd important nucleic acid in cells?
  12. Qualities of DNA Replication
    • -Semiconservative (Meselson & Stahl)
    • -DNA polymerase + host of other proteins
    • -Accuracy & Speed
    • -S phase (Eukaryotes)
  13. DNA Replication Requires?
    • -Strand separation
    • -DNA polymerases
  14. Qualities of DNA Polymerases?
    • -5'-->3' Synthesis
    • -High Fidelity
    • -Accuracy, Speed
  15. Contributions to Fidelity
    • -Polymerase Selectivity
    • -Proofreading
    • -Mismatch Repair
  16. DNA Polymerase Activity
    • -5'-->3' polymerase activity
    • -5'-->3' exonuclease, removal of primer
    • -3'-->5' exonuclease, proofreading activity
  17. DNA Initiation
    • -Bidirectional
    • -Negative supercoiling allows binding of dnaA protein which unwinds/synthesizes primer
    • -dnaB & dnaC: bind dnaA to bend/open the helix
  18. RNA Primer for DNA Synthesis
    • -Required
    • -Primase (RNA polymerase) synthesizes an RNA primer which is ~5 nt long
    • -DNA polymerase III can now begin DNA synthesis
    • -This primer is removed by DNA polymerase I (5'-->3' exonuclease activity)
  19. Okazaki Fragments
    • *1-2000 nt prokaryotes
    • *~200 nt eukaryotes
  20. DNA Ligase
    -Requires terminal phosphate group
  21. Topoisomerases
    • -Unwind and wind DNA
    • -Introduces transient (not permanent) strand breaks to relax DNA
    • -Type I: single strand break
    • -Type II: double strand break (known as DNA gyrase in prokaryotes, hydrolyzes ATP)
  22. Ciprofloxacin & Novobiocin
    • -Antibiotics
    • -Inhibit DNA gyrase but not eukaryotic topo II
  23. Captothecin
    • -Inhibits topo type IB (eukaryotic topoisomerase) by stabilizing enzyme-DNA intermediate
    • -Anticancer agent since nicked DNA can't replicate
  24. DNA Replication in Eukaryotes
    • -Autonomous replication sequence (ARS)
    • -8 Proteins, 11 Base Pair Consensus Sequence
  25. Sliding Clamp Theory
    • -Asymmetrical
    • -Processivity (Allows DNA pol to stay on DNA & not fall off)
    • -Proofreading (3'-->5' exonuclease activity)
  26. Telomeres
    • -Tandem Repeats
    • -Hexanucleotide sequence
    • -Species specific
    • -TTAGGG for humans
    • -On eukaryotes

    • -Free ends of chromosomes present unique problem
    • -3' end of lagging strand

    -Have telomerase (reverse transcriptase) which contains an RNA molecule & species specific
  27. Telomerase in regards to Cancer
    • -Active in cancer cells
    • -Keeps telomeres long
    • -Drugs target telomerase by stopping tumor cell proliferation, thus shortening cancer cells telomeres
  28. Telomerase in regards to Aging
    • -Telomeres shorten with time
    • -Damaged skin, blood vessels, retinal cells can be prolonged by introducing telomerase
  29. Qualities of Transcription
    • -Gene Expression
    • -Regulate mRNA to regulate gene expression
    • -On/off, Rate, Stability, Amount of specific transcripts
  30. Transcription Initiation
    • -Sigma subunit (prokaryotes) of RNA polymerase recognizes a promoter region (no primer needed)
    • -First phosphodiester bond formed
    • -ATP or GTP (purine triphosphate) base pairs to template at +1 site
    • -Consensus sequences: homologous sequence with same function
    • -Prokaryotes: -35 sequence, Pribnow box
  31. Template Strand
    -Non-coding strand
  32. Nontemplate
    -Coding strand
  33. Transcription Elongation
    • -Transcription bubble around 17 bases
    • -RNA synthesized from template (noncoding strand)
    • -No proofreading
  34. Transcription Termination for Prokaryotes
    • -RNA hairpin followed by U-rich region (structural/metabolic genes)
    • -Rho protein pulls RNA from template strand (ribosomal genes)
  35. Transcription Termination for Eukaryotes
    • -No strong termination signal
    • -100 nt downstream
    • -Processing determines final 3' end
  36. Bacterial RNA Polymerase
    • -1 RNA polymerase that transcribes all genes
    • -Primase (specialized RNA polymerase which makes RNA primer needed in DNA synthesis, not involved in transcription)
    • -Rifampicin (inhibits initiation of RNA synthesis)
  37. Eukaryotes RNA Polymerase
    -3 types

    • -Type I (nucleolus)
    • -Type II (mRNA)
    • -Type III (tRNA)
  38. RNA Polymerase Functions
    • -Scans for DNA initiation sites
    • -Unwinds short stretches of DNA (17 bp)
    • -Selects nucleotides
    • -Catalyzes phosphodiester bond formation at +1
    • -Interacts with regulatory proteins
  39. Transcription in Eukaryotes
    • -Different promoters
    • -Upstream regions have enhancers
    • -Extensive processing of eukaryotic RNA
  40. Polycistronic vs. Monocistronic
    Makes several proteins vs. One protein
  41. Eukaryotic Promoter Sequence
    • -Cis elements: promoter or enhancer sequences on DNA
    • -CAAT box, Hogness (TATA) box: These sequences are recognized by RNA pol II

    -Trans elements: bind to cis elements
  42. Processing of mRNA
    • -Methylated G nucleotide (5'-5' linkage)--> Cap
    • -Introns are removed
    • -Poly A tail

    -100-200 residues
  43. "Split" Genes
    • -Introns intervene between exons
    • -Introns varies slightly
    • -Exons range from 45-249
    • -Accurate splicing required
  44. RNA Splicing
    • -Spliceosome: primary transcript + snRNP's
    • -Small nuclear ribonucleoproteins (snRNPs): proteins + small RNA's (snRNA)
    • -Lariet: excised intron

    -siRNA: small inhibitory RNAs-transcribed from some of that junk DNA areas
  45. Actinomycin D
    • -Antibiotic
    • -Inhibits RNA synthesis
    • -Intercalates in dsDNA, prevents DNA from serving as a template
    • -Works in bacteria and eukaryotes
  46. Alpha-Amanitin
    • -Toxin in poisonous mushrooms
    • -Inhibits Eukaryotic RNA polymerase

    • -Type I (nucleolus) --> insensitive, can't block
    • -Type II (mRNA) --> very sensitive, strongly inhibited
    • -Type III (tRNA) --> inhibited only at high concentration
  47. Rifamycin & Rifampin
    • -Changes RNA pol conformation so it can't initiate RNA synthesis
    • -Doesn't bind to eukaryotic polymerase, only bacteria polymerase
    • -Used to treat tuberculosis
  48. Ribosomes in Prokaryotes and Eukaryotes
    • -Eukaryotes have larger ribosomes
    • -Both eukaryotes and prokaryotes have large and small subunit
  49. Ribosomal RNA
    • -Essential for protein synthesis
    • -Cleavage in rRNA abolishes protein synthesis
    • -P site sequence is conserved
    • -rRNA needed for peptide bond formation
    • -Antibiotics interact with rRNA
  50. How is tRNA activated?
    • -Amino acid esters are activated intermediates in protein synthesis
    • -2 ATP required to charge the tRNA
    • -Aminoacyl tRNA synthetase: enzyme for each amino acid
  51. Aminoacyl-tRNA Synthetase
    • -Increases fidelity of protein synthesis
    • -Highly selective
    • -Corrects own erros by hydrolyzing incorrect aminoacyl-adenylate
  52. Codon Recognition
    • -AA not involved
    • -Codon (mRNA) matches with Anticodon (tRNA)
  53. Initiation of Protein Synthesis (Translation)
    • -AUG
    • -5'-->3'
    • -First tRNA enters P site

    • -GTP hydrolyzed
    • -Initiation factors required
  54. Initiation of Translation (Eukaryotes)
    -mRNA binds to small ribosomal subunit

    • -5' cap recognition
    • -Scans to first AUG (P site)
    • -Methionine (First AA)
    • -Greater than 12 eIFs
  55. Initiation of Translation (Prokaryotes)
    • -Shine-Delgarno sequence (purine rich) indicates AUG start
    • -Formyl-Met (First AA)
    • -3 IFs
  56. Peptidyl Transferase
    • -Catalyzes formation of peptide bond
    • -Does so by nucleophilic attack
    • -Energy from incoming AA-tRNA
    • -Activity catalyzed by A residue of 23S rRNA
  57. Translocase
    -EF-G (Translocase) catalyzes the movement of peptidyl-tRNA out of the A site
  58. Termination of Translation
    • -Release Factors read stop codons (UAA, UAG, UGA)
    • -NO tRNA for stop codons

    • -Release factors alter specificity of peptidyl transferase
    • -Water used as an acceptor
  59. Puromycin
    • -Prokaryotes & Eukaryotes
    • -Analog of aminoacyl-tRNA
    • -Premature chain termination
  60. Erythromycin
    • -Prokaryotes
    • -Binds irreversibly to large subunit (50S)
    • -Inhibits translocation
  61. Streptomycin
    • -Prokaryotes
    • -Binds to small subunit (30S)
    • -Inhibits initiation, causes misreading of mRNA
  62. Diptheria Toxin
    • -Inhibits translocation
    • -Blocks translocase by ADP-ribosylation of EF2 catalyzed by A-fragment of toxin
  63. Gene is expressed
    When transcribed
  64. Constitutive expression
    Always transcribed
  65. Regulated expression
    Modulated expression (up or down)
  66. Regulation of Gene Transcription
    • -Lac operon
    • -Tryptophan attenuator
  67. Operon
    -A coordinated unit of gene expression

    -Consists of: Regulator genes, Operator sites, Structural genes
  68. Z, Y, A Gene
    • -Z gene: Beta galactosidase (Lactose--> Glucose + Galactose)
    • -Ygene: Permease
    • -A gene: Transacetylase
  69. No Lactose
    -Lac repressor binds to operator site and prevents transcription
  70. Presence of Lactose
    -The inducer binds to the repressor which prevents it from binding to the operator so the genes can be transcribed

    • -When glucose is absent and lactose is present, the lactose with convert to 1,6 allolactose (inducer)
    • -It binds to the repressor and inactivates the repressor
    • -1,6 allolactose=Alpha-1,6 linked galactose + glucose
    • -RNA polymerase binds to the promoter now and transcription occurs
  71. Catabolite Repression in the Lac Operon
    • -When glucose concentration is low, cAMP increases
    • -cAMP binds to CAP (Catabolite Activator Protein)/CRP (cAMP Response Protein)
    • -This stabilizes the RNA polymerase and enhances transcription 50X
  72. Lactose and Glucose Present
    • -Low cAMP levels
    • -CAP doesn't bind polymerase
    • -Little/no transcription of lac operon genes
  73. Lactose but no Glucose
    • -Inducer binds repressor
    • -Repressor falls off and becomes inactive
    • -cAMP levels are high when glucose is not present
    • -cAMP binds to CAP and stabilizes polymerase
    • -Transcription occurs
  74. Tryptophan Operon
    • -Encodes 5 enzymes that convert chorismate into tryptophan
    • -Contains 14 AA peptide (with 2 Trp next to each other) + Untranslated attenuator sequence
  75. High Trp
    • -High levels of Trp are available, ribosome passes quickly and termination turn forms
    • -Transcription is blocked because there are high levels of Trp readily available
  76. Low Trp
    • -Low levels of Trp causes the ribosomes to stall at Trp codons, the alternative turn forms, which prevents formation of termination turn
    • -Transcription occurs
  77. Post-transcriptional Regulation
    • -Other attenuate operons include:
    • -Threonine, Phenylalanine, Histidine
  78. Are genes organized in Operons?
  79. Positional Information
    -Cells can signal each other and modify gene expression depending on position
  80. Histone Code Hypothesis
    • -Specific combinations of modifications help determine chromatin configuration & influence transcription
    • -Chromatin remodeling
  81. Ways to Alter Genes Available for Transcription
    • -Methylation of DNA (Deactivates it)
    • -Gene Rearrangement (Ig)
    • -Gene Amplification (Response to stimuli)
  82. Heterochromatin vs. Euchromatin
    • -Heterchromatin: Condensed, little or no gene expression
    • -Euchromatin: Open, transcriptionally active

    • -Normal chromatin blocks TFIID & Pol II from associating with DNA
    • -Transcriptionally active genes show DNase I hypersensitivity
  83. Acetylation of Histones
    • -Transcription occurs
    • -Lysine (+) residues on histones accept acetyl groups and decreases affinity of DNA to histones
  84. Deacetylation of Histones
    -Repressor proteins have deacetylase activity
  85. Proteins Regulating Transcription (Trans-elements)
    • -General Transcription Factors (Initiation)
    • -Activators & Repressors (Regulation)
  86. Enhancers (Regulating Eukaryotic Gene Expression)
    • -Modulate activity of RNA polymerase (Bind Transcription Factors)
    • -Position & orientation independent
  87. Silencers (Regulating Eukaryotic Gene Expression)
    • -Control regions of DNA
    • -Area where TF bind and represses by binding to repressors
  88. General Transcription Factors (Basal Factors)
    • -Pre-initiation complex
    • -Basal levels
    • -TFIIA-TFIIH (Each have specific function, TATA & promoter recognition, Pol II & TF recruitment, Bind sequentially, 1-12 subunits, 15-250 kDa)
  89. Homeotic (Hox) Genes
    • -Transcription factors that bind DNA
    • -Position-specific differentiation & body segmentation during development
    • -Wide variety of organisms
    • -Homeobox: 180nt semiconserved sequences that codes for helix-turn-helix binding domain (homeodomain)
  90. Steroid Hormones
    • -Transcription Factor
    • -Derived from cholesterol
    • -Alters PATTERN of expression rather than individual gene
    • -Receptors bind to steroid hormones/retinoids/thyroid hormone
    • -Conformation change allows binding of co-activator

    • -Steroid binds to the hormone, causes conformational change & uncovers Zinc finger DNA binding domain
    • -The complex interacts with GRE (regulatory DNA sequences)
    • -The complex interacts with co-activator proteins and controls transcription of targeted genes
  91. Post-transcriptional Regulation
    • -Alternative Splicing (Allows diversity, many proteins from a single gene is made)
    • -mRNA Stability (Signal near 3' end, AU rich leads to degradation, especially in regulatory proteins, growth factors, transcription factors)
    • -RNA Editing
    • -Regulation of Translation
  92. Endogenous Mutagenesis
    • -Depurination
    • -Oxidation/Free Radical
    • -Errors in Replication
  93. Exogenous Mutagenesis
    • -Ionizing radiation resulting in free radicals
    • -Alkylating
    • -Nitrous acid=deamination
    • -Ethidium bromide=intercalation
    • -Ultraviolet
Card Set
Biochem Exam #1
Biochem Exam #1