exam 3 class notes

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exam 3 class notes
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  1. Gene
    activity is like a dimmer switch for a light never fully on/off
  2. Transcription
    • = gene expression
    • DNA --RNA polymerase--> RNA
  3. RNA polymerase subunits
    • 2 alpha subunits
    • 1 beta subunits
    • 1 beta subunits
    • = core enzymes
    • sigma factor
  4. RNA polymerase
    holoenzyme
  5. sigma factor
    • help identify start points ( prometers)
    • provide a mechanism for RNA  pol. binding to DNA
  6. What is prometer?
    • start location for transcription
    • -consists of 2 major regions
    • TATAAT = -10 start of first base in transcription
    • TTGACA = -35
  7. Location in transription
    where a sigma factor binds
  8. Different types of sigma factors
    • sigma 74 = vegetative phase
    • sigma 54 = n- stress
    • sigma 70 = heat shock response
  9. Steps in transcription
    • inittiation = sigma factor > RNA pol holoenzyme
    • > elongation > 5' > 3' (reg,free 3' OH) direction
    • -sigma factor released after initiation  
    • -once  the mRNA is formed it is now available for translation
    • occurs stimultaneous transcription + translation
  10. Transcription error rate
    1-105 bases
  11. DNA polymerization error rate
    1-109 bases
  12. Terminiation
    • 2 possible mechanisms
    • -RHO (protein0 - independant
    • -RHO- dependant
    • RHO - independant -> stem loop structure
  13. The RNA polym interaction w/DNA in the buble?
    termination of RNA synthesis
  14. Rho dependant
    • when a transcript matures (news it full lenght )
    • R protein 'p' - Rho binds to RNA + moves up the transcript + interacts vo/ RNA pol + destabilizing it + causing termination
  15. Rho -
    knows when to bind when RNA pol pauses briefly 
  16. Strenght of promoter
    those regions that bind  sigma factor most effectively are strong --> and yield a lage amount of RNA transcripts
  17. Translation
    • protein synthesis
    • start:--> shine- delaerno sequence ribosome binding site
    • A-G (AGGA) => rich sequence
  18. Ribosome:
    • mRNA = 55, 16,s , 23s
    • mProteins- large subunits 30 and small subunits 20
  19. crytallography
    • of mRNA + their accociated proteins have allowed us to understanf protein synthesis
    • -novel drugs can be designed to interfere w/ ther process
  20. Requirements for protein synthesis
    • 30s, 50s,tRNA
    • every amino acid has a coresponding set of codons
    • the insertion of an amino acid into a growing protein is moderated by the tRNA that has a match VIA, B anti-codon to the corresponding codon on the mRNA
  21. Acessory  proteins
    • initiation factors:
    • -IF ; IF2 ; IF3
    • elongation factors:
    • EF- TU
    • EF - G
    • Termination factors:
    • Release factors
  22. initiation of translation
    • 30s subunit bond by IF3 + IF1
    • IF#; help id the RBS
    • IF1 blocks the site where the incoming formy-met t-RNA will interact
    • IF2- brings in the F-met tRNA w (GTP)
    • once this complex is assembled the large subunit can assoc w/the small subunit
  23. IF3
    help id the RBS
  24. IF1
    blocks the site where the incoming formy-met t-RNA will interact
  25. IF2
    Bringing in the F-met tRNA w(GTP
  26. Visualizing the three sites?
    • A- acceptor
    • P- peptdyl transfer
    • E- exit
  27. Elongation of Translation
    • requires two activities
    • 1) petdyl transferase - large subunit associated activity
    • Allows for peptid bond formation between the 1st Met and the incoming Amino acid in , the A site
  28. Translocation
    • ribosome moves 1-codon over towards the 3' end of the mRNA
    • A)-this activity causes the grwoing polypetides tRNA to be located int the P site
    • b) entry tRNA (the one carrying the F-net) to move into the E (exit) site
    • c) A site to be vacant (so that another tRNA w amino acid can enter)
  29. Termination fo protein synthesis
    • UAA / UGA / UAG (stop codons)
    • -sequences have no matching anti codon tRNA so nothing new will come into the A site
  30. proteins secreted or final destination(termination of protein synthesis) GSP
    • som proetins are secreted or may have final destination outside the cytoplasm
    • -cytoplasmic membrane
    • -periplasm
    • -O.M
    • -ENV
    • so signals present in the newly formed proteins provide for this activity = protein secretion
    • General secret Dry process (pathwyas) GSP
  31. gene regulation
    • regulation of catabolic pathways
    • regulation of anabolic
  32. lac operon pathway (gene regulation)
    catabolic pathway
  33. operon (gene regulation )
    cluster of genes
  34. operon types
    • promoter
    • operator
    • structural genes
  35. Lac operon
    regulates lactose catabolism
  36. lac Z
    beta galactosidase
  37. beta-galactosidase
    brease down lactose into glucose and galactose
  38. lacy
    coding for lac (permease) involved in uptake of lactose into the cell
  39. lac A
    function uknown / transacetylase
  40. repressor (lac I )
    lac repressor
    binds to operator that will block further movement of DNA polymerase turn on/ off mechanism
  41. Inducible operon
    • inducer --> lactose should be present
    • lactose absent --> turn off
  42. lactose present
    • beta-galactosidase
    • lactose = allolactose
    • repressor binds allolactose => repressor inactive
    • in order to turn on it should alwasy bind to the operator
    • lacz--> mRNA --> LacZ
    • lacy --> mRNA --> Lac Y
  43. Regulation of anabolic pathways
    • ex: biosynthesis of amino acid
    • - hisdtidine operon
  44. 3 types of enzymes (regulation of anabolic pathways
    • all 3 always regulated
    • inducible enzyme
    • repressible enzyme
    • -WOT essential
    • constitutive essential
    • -always regulated all three
  45. Inducible
    Lac operon
  46. Repressible
    Histidine
  47. Histidine operon
    function when is needed
  48. When hestidine is in adequate level?
    Attenuation --mechanism--> to prevent our production of histidine
  49. Translation
    • ribosomal operon (rrn operon )
    • Ribosome --> rRNA ribosomal proteins
  50. Catabolite repression (global regulation )
    • glucose
    • lactose
  51.  CAMP (global Regulation)
    • cyclic AMP
    • is an activator
  52. CRP
    catoblite repressor protein
  53. Glucose levels
    • when glucose levels high CAMP goes down
    • glucose increase CAMP decrease
  54. CAMP should bind to?
    • CAP
    • binding enhances transcription
    • cannot bind to CAP if CAMP is low
    • complex binds to promoter leading to transcription of genes involved n lac operon
  55. Chemical mutagens
    5-bu- structural
  56. alkylating agents
    CH3--> guanine
  57. thymine dimers (physical mutagens )
    • A=T
    • T=T
  58. DNA repiar ( physical mutagens )
    • dark and light
    • light photolyse
    • ligase = joining
    • endo nuclease- within DNA
  59. Transposable elements ( bioligical mutagens)
    • jumping genes
    • barbara mcclintock
    • in maize
    • eukaryotes adn prokaryotes
  60. insertion sequence ( bioligical mutagens)
    CCAT = TACC (palindrome)
  61. Transpons
    anibiotic resistance
  62. Plasmid
    • e.coli stx gene
    • bacteriocins
    • -in plasmid capable of killing
    • ex. e.coli plamids
    • killing closely related organisms
    • colicins
    • resistance plasmids - R-factors
  63. Genetic exchange
    • vertical / horizintal / lateral
    • 3 types
    • -transformation
    • -conjugation
    • -transduction
    • merozygote: hologous recombination
  64. Transformation
    • no direct contact
    • uptake- extracellular DNA
    • competence - closely related bacteria
    • natural /artificial - creates pores in the cell membrane
  65. Conjugation
    • -direct contact
    • F plasmid: F+ F+
    • F+--->F-
    • conjugative plasmid --> F plasmid
  66. Transduction (generalized )
    • bacteriophage - mediator
    • transduction
    • - generalized
    • -specialized
  67. Generalized transduction
    Bacterial DNa- is incorporated to phage ( transducing particles)
  68. Generalized vs specialized
    • Generalized:
    • -all regions  of of chromosome transduced
    • -only chromosomal DNA ( i.e no phage DNA in tranducing particles )
    • Specialized:
    • -bacterial and phage DNA
    • -only specific regions of chromosome near attachment site
    • -transducing particles contains ( phage DNA as well as bacterial DNA)
  69. Viruses
    • -nucleic acid
    • -protein coat (capsid)
    • capsomers sub units of capsules can effect any organism
    • virus-> poison
  70. David baltimore
    • noble prize  Z more viruses gives cancer
    • -reverse transcription
  71. Baltimore classification 6-7 groups  ( viral classification )
    • based on virus genome
    • double stranded DNa viruses
    • Single stranded DNA viruses
    • double stranded RNA
    • +single stranded RNA - coding
    • -single stranded RNA - template
    • Retro viruses (reversre transcription)
    • DNA Reverse (transcribing viruses 0
  72. double stranded DNA viruses
    • traditional gene expression
    • DNA--> mRNA--> Protein
    • need hoste
    • viruses: obligate intracelular pathogen
    • -depend on the host enzyme
  73. Single Stranded DNA
    • host DNA polymerase
    • convert single stranded DNA to double strande DNA with help of polymerase,( host RNA polymerase)
  74. Double stranded RNA
    • viral RNA dependent RNA polymerase
    • mRNA
  75. + single strand RNA
    act as a coding strand during an infection the RNA acts like mRNA and can be translated by hosts ribosomes
  76. single stranded RNA
    • converted into (+SS) RNA
    • act as a template doing transcription to make a complementary ( +) sense mRNA
  77. Retroviruses
    • HIV
    • type of +ssRNA 
    • -reverse trancriptase TNa become DNA
  78. DNA reverse transcribing viruses
    • reverse transriptase
    • DNA --> RNA 
    • DNA <--- RNA
  79. structure (viruse)
    • envelope -lipid layer
    • naked -- no envelope
    • naked and evnelop = verions
    • virion = mature viruse
    • spikes- glycol proteins help in attachment in host cells
  80. Viral capsid
    complex Viruses bacteriophage
  81. viral envelope
    organic solvents --> ether
  82. Viral life cycle
    • 5 steps
    •  adsorption
    • penetration and cuncoating
  83. Phage counts
    • plaque- zone of inhibiyion on bacterial lawn due to viral attack
    • pfu/ml
    • pfu/ml = average 3 of plawue X dilution facter
  84. Replication pathways
    • lytic (virulent phages) kill host
    • lysogenic (temperate phage ) no killing
  85. virulent phage life cycle
    • life cylce of a virulent phage
    • last step: lysis of bacterial cell
  86. life cycle of a temperate phage
    • viral DNa is incorporated to bacterial DNA
    • --> prophage
  87. Switching from lysogenic to lytic
    • stravator
    • radiations
  88. Animal viruses
    • whole animals
    • embryonated chicken eggs
    • cell cutlre
  89. Replication of animal viruses
    • envelope fussion (endocystosis )
    • phagocytosis
    • injection of nucleic acid
  90. Viral recongition of host cell
    • canyon--> dipressions
    • viral penetration
    • a)
    • b)phagocytosis
    • c)injection
  91. uncoating and synthesis
    • DNA viruses mRNA
    • replication and gene expression by dsDNA viruses budding
  92. Retro viruses
    • +ssRNA
    • RNA --> DNA reverse transcriptidase
    • ex: HIV
    • 3 enzyme
    • -intergrase
    • -protease
  93. HIV life cycle
    • enveloped
    • 1. binding and fusion
    • 2. reverse transcription
    • 3. integration
    • 4. transcription (normal0
    • 5. assembly
    • 6. budding
  94. gp120 HIV viruse
    binds and fuses to CD4 receptor cell ( macrophage0
  95. (+)ssRNA
    • Dna Reverse transcription
    • using reverse transcriptase in the viral genome
  96. Viral DNA
    is integrated into the host DNA using viral enzyme (integrase)
  97. Provirus
    HIV and human DNA
  98. Normal gene expression ( transcription)
    • viral proteins
    • leave the cells ( budding )
  99. Rho factors
    • TaTa box
    • -tRNa -clover leaf model
  100. Protease
    will cute long chains of proteins and make short peices of DNA then assebly with the nucleic acid

    Budding
  101. Drugs that target retovirus enzymes
    Azidothymidine - druge for retroviruse - reverse transcriptase
  102. Viroids
    • single stranded
    • virods and prons - subviral particular no protein
    • infect plants -> potatoes, tomatoes , and peahes
    • potatose spindle tuber viroid --> potato spindle disease
    • Hepatitis D --> by viroid
  103. Prions
    • no nucleic acid
    • fatal
    • PrP - function of normal Prp unknown
    • Prp- misfolded
    • infected animals PrP gets misfolded
    • blanks =sheep / humans
  104. bacterial diversity 3 domains
    • 3 domains
    • -euaryia
    • archea
    • bacterial
  105. Scientific nomenclature
    genus (first)
  106. stromatolites
    tell how old microorganism is
  107. Wittaker, 5 kingdoms
    • plantae, animals
    • funi moneroa
    • protists
  108. cal woese
    divided it into three domains
  109. endosybiotic theory
    orgnaism living within another -lynn margulis
  110. DNA hybridization
    • heat DNA strand to denacture DNA strand
    • 95-98C annealing (cooling)
    • 45-60( two strand together
    • 100% hydridization = identical or not
  111. macromolecule sequencing
    rRNA - say organisms are related or not
  112. Domain Archea
    • 3phyla
    • crenoachaeota
    • class thermoproteli
  113. Euryarchaeota
    • methanogens
    • extemphalophiles
    • theroacids
  114. mathogens
    • produce mehtane CH4
    • -anerobic conditions
    • ex: rumen of cow
  115. extremehalophiles
    • saline 10% NaCl
    • ex: halobacterium
  116. thermoacidophiles (extreme thermophiles
    • high temp
    • low ph
    • sulfolobus ( H2SO4) low pH hogh acidic conditions
  117. B10 Cyanobacteria
    • oxygenic photosynthesis
    • nitrogen fixation
    • oldes microorganism
    • heterocyst - N2 fixation
    • -notic
    • anabaena
    • blue green algae
    • azikka + anabueria
  118. alphaproteobacteria
    •  3 important bacteria
    • 1. agrobacterium
    • 2. acetobacter 
    • agrobacterium
    • crown gall in plants
    • -genetic eginering T-DNA
    • acetobactor vinegar
    • rhizobium
    • N2 fixer
    • symbiotic accociation with leguminous plants
    • rood nodes
    • meso /sino/ bradylhizobium
  119. alphaproteobacteria
    cloroplast -cyanobacteria
  120. betaproteobacteria
    whooping cough
  121. Gammaproteobacteria
    • enteric
    • -e.coli
    • -shigella
    • yersna pestis -- bubonic plaque
    • indole - true
    • typrtophanase --> indole
  122. pseudomonas eruginosa
    • cystic fibrosis burns
    • environmentally
    • psedomona putida - bioremedation
  123. Epsilonproteobacteria
    • camphylo bacter - intestinal disease
    • helicobactor pylori - cause peptic ulcers

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