Lecture 21: Eubacterial Transcription, DNA-binding Assays

Question 1

Prokaryote vs. Eukaryote transcription processing

Answer 1

• eukaryote - more complex process
  
  1. transcription
  2. add 5’ cap and poly a tail
  3. rna splicing
  4. export out of nucleus
  5. translation
• Prokaryote
  
  1. transcription
  2. translation

Question 2

Requirements for RNA synthesis

Answer 2

• DNA template (3’ to 5’)
• Enzyme - RNA polymerase (processive - stay binded for long time)
• Ribonucleotides (G, A, U, C)
• Start signal - Promoter (usually starts with Purine)
• Stop signal
  *

Question 3

Bacteriophage T7 RNA polymerase

Answer 3

• single subunit
• right hand claw
• promoter: TAATACGACTCACTATAG
  
  • transcription start point: +1
  • more consensus with this sequence makes stronger promoter
• terminator:
  
  • loop is gc rich and after loop is AU rich

Question 4

3 classes of RNA in Prokaryotes

Answer 4

1. Ribosomal RNA: structural component of ribosome
2. Transfer RNA: decodes mRNA
3. Messenger RNA: carries information for protein

• in order of most to least common (rRNA is most common)

Question 5

Prokaryotic RNA Polymerase (function, parts, )

Answer 5

• single, multisubunit enzyme
• transcribes all 3 RNA
• transcription can occur from either strand
  
  • always 5’ to 3’
  • upstream: 5’ and downstream: 3’
• holoenzyme
  
  • core enzyme
    
    • 2 alpha subunits: recognizes UP element for strong binding
    • beta: catalytic core
    • beta prime: recognizes template strand
    • omega: recognizes promoter elements
  • sigma subunit: binds promoter then elongates 5-10 nucleotides then sigma dissociates
    
    • most common cofactor in e. coli
    • binds to -35 and -10 promoters

Question 6

Consensus sequence (promoter for RNA polymerase)

Answer 6

• sigma subunit contacts -10 and -35
  
  • -10: Pribnow box (AKA TATA box)
    
    • contacted by domain 2
  • -35:
    
    • contacted by domain 4: helix-turn-helix motif
• alpha subunit contacts UP element
  
  • AT rich

Question 7

Transcription initiation process

Answer 7

1. Binding (closed complex)
2. unwinding (Open complex
   
   • energy independent
   • conformational change in RNA polymerase
3. Synthesis begins at +1
   
   • TSP - transcription start point
   • TIS - transcription initiation site
4. forms stable ternary complex
   
   • promoter clearance
   • sigma factor disengages
   • progresses to elongation stage
5. Elongation
   
   • template strand-directed nucleotide polymerization by base pairing
   • 50 nucleotides per second at 37C
   • Gre and Nus factors help proofreading and elongation by RNA polymerase during elongation
   • elongation bubble: ~17 bp bubble, ~8bp RNA DNA hybrid
6. Termination by signals within RNA sequence
   
   • ​​Rho dependent/independent termination

• release polynucleotide
• polymerase dissociates from template

Question 8

Rho independent termination of transcription

Answer 8

• forms hairpin
• release RNA chain since AU base pairs dissociate easily

Question 9

Rho dependent termination of transcription

Answer 9

• Rho recognizes rut sites (~40 nt, C- rich, mainly ss)
• Does not bind RNA that is being translated
• ATP-dependent helicase
  
  • unwinds RNA from DNA template
  • dissociates RNA poly

Question 10

Electrophoretic Mobility Shift Assay (EMSA)

Answer 10

• gel shift
• used to see if protein binds to DNA
• electrophorese in non denaturing polyacrilamide gel
  
  • labeled oligonucleotide probe
  • protein and probe
  • specific antibody and protein and probe

Question 11

DNase Footprinting

Answer 11

1. end-labeled DNA fragment - add purified protein to one sample, leave the other as is.
2. partial DNase I digestion
   
   • won’t cleave where the protein is binded
3. on x-ray film, there will be no footprint where protein binded