Transcription

Question 1

describe the process of transcription

Answer 1

• RNA polymerase catalyzes the synthesis of an RNA molecule in the 5’ to 3’ direction along the 3’ to 5’ template strand of the DNA
• Ribonucleoside triphosphates are the RNA precursor molecules used for RNA synthesis

Question 2

what is the name of the DNA strand used for transcription?

Answer 2

Template or antisense strand

Question 3

What is the name of the DNA strand not used for transcription?

Answer 3

Sense strand (same sequence as RNA but with U instead of T)

Question 4

compare DNA synthesis with RNA synthesis

Answer 4

• RNA synthesis
  
  • RNA pol
  • NTPs precursor
  • no primer needed
  • uracil pairs with adenine
• DNA synthesis
  
  • DNA pol
  • dNTP precursor
  • primer needed for initiation
  • thymine pairs with adenine

Question 5

name the 4 different types of RNA molecules

Answer 5

• mRNA: encodes the amino acid sequence of a polypeptide
• tRNA: brings amino acids to the ribosome during translation
• rRNA: combines with ribosomal proteins to form ribosome
• snRNA: combines with certain proteins and is involved in RNA processing (mRNA splicing) and gene reg. in eukaryotes

Question 6

what is the RNA pol in prokaryotes?

Answer 6

• Single RNA pol composed of the core polymerase + sigma factor
  
  • sigma factor enables RNA pol to recognize promoters

Question 7

what is the RNA pol in eukaryotes?

Answer 7

• RNA pol I: rRNA genes
• RNA pol II: all protein-coding genes (mRNA), some snRNAs and microRNA (miRNA)
• RNA pol III: tRNA genes and some snRNAs

Question 8

name the regions of the prokaryotic gene

Answer 8

• Promoter: located upstream of the RNA coding sequence and ensures the proper location of transcription initiation
• RNA coding sequence: the DNA sequence that is transcribed into RNA
• terminator: a sequence downstream of the RNA coding sequence and specifies where transcription will stop

Question 9

where are the prokaryotic promoter sequences located?

Answer 9

• -35 and -10 bp from the transcription start site
• Variation within these sequences results in variation in the binding ability of RNA pol and can affect the rate of transcription

Question 10

describe initiation and elongation of the RNA transcript in proks

Answer 10

1. RNA pol (holoenzyme - core RNA pol + sigma factor) binds to -35 and -10 regions
2. DNA molecule is unwound by approximately 17 bp in the -10 region
3. After 8 or 9 ribonucleotides have been polymerized the sigma factor dissociates from the core enzyme
4. Polymerization proceeds as RNA pol unwinds DNA molecule
5. Transcription is terminated once termination sequence is reached

Question 11

explain rifampin

Answer 11

• An antibiotic
• Inhibits initiation of transcription by blocking the formation of the first phosphodiester bond by binding to prokaryotic RNA pol
• RNA pol of eukes is not affected
• Used in tuberculosis treatment

Question 12

name the 5 proteins (we need to know) that are involved in transcription initiation in eukaryotes

Answer 12

1. TATA box binding protein
   
   1. Subunit of TFIID
   2. Binds to TATA box of gene promoter
2. TFIID
   
   1. causes a distortion in the DNA helix allowing the recruitment of other transcription factors
3. TFIIB
   
   1. Involved in RNA pol interactions
   2. start site recoginition
4. TFIIH
   
   1. contains a DNA helicase to unwind DNA
   2. activates RNA pol by phosphorylation
5. TFIIE
   
   1. Involved in positioning RNA pol

Question 13

describe the transcription initiation in eukaryotes

Answer 13

1. The most common euk promoter sequence is located at -25
2. TBP/TFIID bind to the promoter at the TATA box
3. TFIIA and TFIIB binds to promoter (recruitment and stabilization)
4. RNA pol, TFIIE, and TFIIH are recruited to the promoter and transcription initation site
5. TFIIH unwinds the DNA and activates RNA pol by phosphorylation
6. TFIIH phosphorylates RNA pol activating it for transcription
7. Most transcription factors are then released from the basal transcriptional machinery prior to transcription initiation

Question 14

what is the basal transcriptional machinery?

Answer 14

• Made up of RNA pol II and TFs
• Basal transcriptional machinery is sufficient for only a low level of transcription
• For increase levels of transcription or tissue specific transcription, the binding of an activator and adaptor molecule is required

Question 15

describe the mushroom Amanita phalloides

Answer 15

• α-amanitin, found in Amanita phalloides (death
  cap mushrooms), inhibits RNA polymerase II.
• Causes severe hepatotoxicity if ingested.
• Treatment
  
  • GI decontamination
  • High dose penicillin (inhibits amanitin activity in liver)
  • Liver transplant

Question 16

what inhibits bacterial gyrase from creating negative supercoils?

Answer 16

• Antibiotics
  
  • Courmarins (novobiocin)
  • Quinolones (nalidixic acid and ciprofloxacin)

Question 17

what causes intrinsic (aka rho-independent) termination of transcription?

Answer 17

• Palindromic regions form hairpins varying in length from 7-20 bps
• The stem loop structure includes a G-C rich region and is followed by a run of U residues
• OligoU binding to the DNA template is very weak allowing separation of RNA from template

Question 18

what causes extrinsic termination (aka rho-dependent)?

Answer 18

• ρ (Rho) factor attaches to nascent RNA at C-rich region behind the RNA pol
• Rho moves along following the RNA pol in the 5 to 3 direction
• RNA pol pauses at termination site allowing rho to catch up
• Rho unwinds the RNA - DNA hybrid duplex forming the transcription bubble
• Newly synthesized RNA is released

Question 19

describe the function of Actinomycin D

Answer 19

• binds tightly to ds DNA
• inhibits transcription and replication in both proks and euks
• primarily used as an anti-cancer drug (inhibiting DNA replication and cell division)
• not normally used as an antibiotic

Question 20

describe function of TFIID

Answer 20

• causes distortion in the DNA helix allowing recruitment of other transcription factors

Question 21

describe the function of TFIIB

Answer 21

• involved in RNA pol interactions
  
  • start site recognition

Question 22

describe the function of TFIIH

Answer 22

• Contains a DNA helicase to unwind DNA
• activates RNA pol by phosphorylation

Question 23

describe the function of TFIIE

Answer 23

• involved in positioning RNA pol