Basics of Transctription

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Basics of Transctription
2012-10-21 00:31:10

start of test 2 material
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  1. Transcription occurs in the ______in eukaryotes and translation in the _____.
    Nucleous; cytoplasm
  2. In prokaryotes transcription and translation occur.....
    Simultaneously, once the mRNA is made translation begins
  3. Where does transcription take place in prokaryotic cells?
  4. RNA polymerase synthesizes a _____ copy of _____
  5. What forms the protein complex at the start site of transcription?
    RNA pol and transcription factors
  6. Genomic DNA is....
    Double stranded and encodes multiple genes
  7. Contains all info for correct regulation, specifies start site of transcription
  8. What does precursor RNA make
    • mRNA and translated it to proteins
    • other RNAs (rRNA, snRNA)
    • involved in RNA processing, transclation, mRNA stability and transcriptional control
  9. Proteins and mRNA can _____ transcription and ______ to the final product
    regulate; maturation
  10. Genes are orginized into
  11. mRNA is polycistromic and gives rise to...
    multiple proteins
  12. Controlled by proteins that activate or repress transcription
  13. E. coli RNA pol....
    • acts like a zipper and unwinds DNA (17bp)
    • non-template strand displaced outside
    • RNA synthisized inside
    • covers about 35 bps
  14. ____ removes negative supercoils and _____ removes positive supercoils
    Topo I; Topo II
  15. RNA pol subunits
    • 2 alphas
    • beta
    • beta'
    • omega
    • sigma factor
  16. The BB' contains the _______ and has _______ activity
    Acitve site; enzymatic
  17. RNA pol
    • Can bind and catalyze RNA but has no specificity
    • Can't recognize promoter
    • Recognition by the sigma factor
  18. Regulates binding to the promoter, has multiple units, and may act to stabilize the enzyme
    Sigma factor
  19. Interacts with other TF
  20. Important Sigma factors
    • 70: normal; most abundant
    • 32: heat shock
  21. E. coli propoter contains:
    • region @ -35 and +10 bound by sigma factor
    • the UP element
  22. Consensus DNA binding sites
    the most common nucleotide in each position in hypothetical binding sequences
  23. Heat shock promoters
    • signa 32 recognized promoters
    • UP is pound by RNA subunit
    • binding sites have different sequences
  24. How does initation start?
    • Pol promoter binds to DNA in the major groove and slides down the DNA or jump from position to position
    • sigma factors binds tightly
  25. Initiation of transcription
    synthesized from the 5' end using the template strand
  26. Promoter Clearence
    • sigma factor no longer interaxts with DNA specifically after synthesis of first 8-9bp
    • ofter released, sometimes noves with pol
    • bubble increased to 17bp
    • RNA/DNA hybrid forms; ~8bp
  27. Rho independent termination
    • string of U's at 3' end weakens duplex
    • hairpin formation, 15-20 bases before end of strand causes pol to fall off
  28. Rho dependent termination
    • no A's in template
    • sometimes forms a haripin
    • Rho binds to the loading site on RNA and migrates to termination site
  29. How doeslactose get into E. coli cells
    transported by lac permerase
  30. Lactose is converted into ______ and ______
    glucose; galactose (sometimes allolactose first)
  31. Lac operon
    • Produces mRNA encoding 3 orf's
    • mRNA controlled by activity of promoter
    • repressor produced from own mRNA and promoter P
    • Repressor binds to O1,O2, and O3 ihibiting lac operon expression
  32. Lac repressor
    • made continuously
    • binds pair of operator sequences and inhibits transcription
    • basel livel of beta gal and lac permease at all times
    • essential for lac operon control
  33. In the presence of lactose 
    • Allolactose is produced and binds to the lac repressor
    • prevent lac repressor from binding to DNA
    • lac promoter expression increades
    • basel expression of lac permease and beta-gal is essential to the control
  34. Negative regulation
    • Repressor bound to the DNA no transcription
    • Modulator bound to repressor away from DNA = transcription
    • Modulator bound to repressor on DNA = no transcription
    • unbound repressor and modulator = transcription
  35. Repressor bound to DNA
    • Binds as a dimer to operator seq
    • 2 pairs associate to form a tetramer
    • DNA forms loops around tetramer with tighest affinity for O3 represor
    • The alpha helix binds in the major groove
  36. Allolactose's conformational change
    withdraws the DNA binding domains preventing DNA binding
  37. Lac promoter in molecular biology
    • Can be put in front of a gene of interest
    • Little/no expression of this gene in the absence of an inducer
    • Transcription is induced by addidion of artificial inducer IPTG
    • Use to drive expression
  38. In a mormal E. coli cell with normal operon....
    • in the presence of allolactoes beta gal with break it down
    • inducer is removed
  39. Weak promoters
    • in the presence of allolactose there is little expression of lac promoter
    • -10 and -35 regions are diverged from the consenses seq and bind the sigma factor weakly
    • no UP seq and low beta gal production
  40. In the well feed state, there is little _____ expression even in the presence of _______
    lac operon; lactose
  41. During starvation
    • lac operon is turned on and lactose is present
    • allows use of lactose as a carbon source when othere perferential sources (glucose) are absent
  42. Starvation activates ______
    CRP (cAMP receptor protein)
  43. CRP
    • binds lac promoter and activates transcription if lactose is present
    • binding is induced by cAMP
    • CRP and cAMP is an example of postive regulation
    • When bound => transcription
    • binds as dimer and change DNA to palimdrome seq
  44. Low glucose, high cAMP
    • cAMP binds to CRP
    • lac repressor released
    • RNA pol activated
  45. High glucose, low cAMP
    • No CRP bound
    • no transcription
  46. Positive regulation 1
    • Activation: CRP bound alone = transcription
    • Inactivation: CRP unbound but cAMP bound = no transcription
  47. Positive regulation 2
    • Activation: CRP bound with cAMP = transcription
    • Inactivation: CRP unbound alone = no transcription
  48. Polymerase I
    • 45S per-ribosomal RNA synthesis
    • occurs in the nucleous
    • nucleolar processing to 18, 5.8, and 28S rRNAs
    • requires snoRNAs
  49. Polymerase II
    • mRNA synthesis, some snoRNAs
    • occure in the nucleoplasm
  50. Polymerase III
    • synthesis of tRNA, snRNA, and 5S RNA
    • ocurrs in the nucleoplasm
  51. snRNA
    small nucleolar RNA
  52. snRNA
    small nuclear RNA
  53. Pol II
    • 10-14 subunits that perform the function of the 6 in bacterial RNA pol
    • interact with other proteins involved in eukaryotic transcription
    • cannot initiate transcription alone
  54. Large Pol II subunit
    • 240 kDa (homoenzyme >500kDa)
    • homologous to B' (second largest is same as B)
    • CTD ontains 7 AA repeat: tyr-ser-pro-thr-ser-pro-ser (repeats ~50x)
    • ser and thr can be phosphorylated
  55. Eukaryotic promoters
    • specifies the start site
    • specifies when (during development) and where (on the gene)
    • regulate transcription of all RNA
    • found 5' of transcription unit
    • start region is internal
  56. Cis-acting promoters
    encode on the same DNA
  57. Trans-action promoters
    action by a factor; anything that acts on the gene to change transcription
  58. Proximal promoter
    • region clsoe to the start site of transcriotion
    • specifie the exact start site
    • binding site of pol and basal TFs
    • contains everthing that allows transcription
  59. Distal Promoter
    • distand (away) form the start site
    • regulates the rate of gene expression
    • may contain enhancers
    • responsible for the postion of pol II
  60. Enhancers
    • element that works up/down stream in an orientation independent manner to regulate transcription
    • the more proteins bound the better
    • changes the structure to affect transcription
  61. TATA box
    • TATAAA consensus (A-T rich region)
    • not alway present
    • mostly found in highly expressed genes
    • binds to TFIID
  62. initiator
    • found near TATA box
    • unwinding site
    • interacts with complexes containing TFIID
  63. downstream promoter & core elements
    • not always present
    • interacts with complexes containing TFIID
  64. Regulatory sequences
    • binds lots of regulatory TF at once
    • often 5' of TATA box but can be anywhere
  65. Pol II has no specific ______ for transcription
    Start site
  66. Pol II activity is controlled by _____and _____ that bind to distant DNA elements.
    basal transcription factors; other TFs
  67. Prokaryotic Promoters
    • recognizes specific DNA seq
    • interacts w/ proteins that bind locally
    • contained w/in ~80bp
  68. TATA binding proteins (TBP)....
    • binds the TATA box and specifies the start site for transcription
    • occurs abot 25 bp downstream of TATA box
    • part of TFIID
    • binds in the minor groove and bends ~80 degrees
  69. TBP binding to DNA
    • binds as a monomer w/ sudo-dimer character
    • covalently bound
    • 2-fold acix of symmetry
    • binds to minor groove - induced fit
    • no inherent directionality
    • beta sheets bind and stabilized by alpha helix
  70. Transciption initiation occurs
    via a closed complex where the factors bind as a chain of events
  71. Open complex
    once all the proteins are bound, DNA start to unwind in the pol unit
  72. Promoter Clearance
    • helicase activity of TFIIH
    • phosporylation of CTD of polymerase
    • TFIID and other proteins left behind
    • TBP taken along with pol
  73. The 5' G-cap is found in ______
    eukaryotes and varies by species
  74. 5' cap
    • protect against 5' degradation
    • added at the start of transcription
    • added to 5' mRNA via 5'-5' triphospate bond
    • GTP is nethylated
    • requires methyltransferase to add adoMet
  75. The primary transcript is the mRNA with ____ and ____ or is not ______
    introns, exons, mature
  76. Primary transcript is considered the _______ product and contains about ________ bp
    first finished, 100,000
  77. Mature mRNA has _____ removed
  78. Splicing is the removal of _____ and joning of _____
    introns, exons
  79. Splicing in mRNA
    • requires small nuclear ribonucleoproteins (snRNPs)
    • contain small nuclear RNAa (snRNA)
    • 5 highly conserved snRNAs
  80. spliceosome assembly
    • uses U1 and U2 to bring the exons together
    • requires ATP to build up RNA complex that removes introns
  81. The Poly A tail _____eukaryotes and ______ prokaryotes
    stablize, destablize
  82. Poly A tail
    • added to the end, not encoded
    • RNA transcribed past the pol A site
    • AAUAAA pol A addition site located 10-30 baseds 5' of mature 3'end
    • GU rich seq 20-30 bases downstream f 3' end
    • large enzyme complex cleaves RNA and adds pol A 80-250 As)
    • doesnt require template