21-22: TRANSCRIPTION

Question 1

one major difference between DNA replication and RNA synthesis?

Answer 1

DNA replication has replication bubble with 2 forks, RNA sythesis has a transcription bubble in one direction

Question 2

the different polymerase enzymes in DNA replication and RNA synthesis

Answer 2

DNA replication uses DNA pol III, RNA synthesis uses RNAP

Question 3

which strand is the template strand in both processes?

Answer 3

DNA replication: both DNA strands at once, RNA synthesis: coding region of genes on the template strand

Question 4

is a primer required?

Answer 4

YES for dna replication, NO for RNA synthesis

Question 5

what is copied in each process?

Answer 5

whole genome in DNA replication, limited segment in RNA synthesis

Question 6

parts of initiation in RNA synethesis?

Answer 6

1. closed complex: RNA Pol binds to the promoter. 2. open complex. 3. initiation of transcription (first nucleotide binds)

Question 7

subunits in the RNAP at initiation?

Answer 7

it’s the holoenzyme: 6 subunits with a sigma factor. very high affinity to DNA but promoter specific

Question 8

how many subunits in the RNAP at elongation?

Answer 8

5. no sigma factor, high non-specific affinity to DNA

Question 9

what do the beta and the beta-prime subunit form?

Answer 9

2 pincers = the active site of RNAP. contains zinc and magnesium. coordinates NTPs and 3 Asp residues

Question 10

what happens and is needed in elongation

Answer 10

all 4 ribonucleoside triphosphates at equal concentration. magnesiu, DNA template, RNAP. rate of 50-90 NTP/sec, 1 error every 10^4 NTP

Question 11

what are the promoter regions and how far apart are they?

Answer 11

-35 and -10, two helix turns apart

Question 12

where does transcription start? and where is the first codon in protein translation

Answer 12

+1, +8 to +13 = ATG

Question 13

what happens in the closed complex?

Answer 13

the holoenzyme binds from -90 to +20

Question 14

what happens in the open complex?

Answer 14

the DNA opens up from -12 to +3

Question 15

when does the sigma subunit detach? and what is it replaced by?

Answer 15

after the addition of 8-9 nucleotides. replaced by protein NusA

Question 16

what are the steps involved in termination? and what are the two mechanisms?

Answer 16

1. phosphodiester bond formation ends. 2 DNA-RNA hybrid dissociates. 3. melted DNA strands in the transcription bubble reassociate. 4. RNA Pol releases DNA. two mechanisms are ‘terminator’ palindrome and the ‘Rho’ protein

Question 17

how does the terminator palindrome work?

Answer 17

the terminator palindrom ends in 3 A bases. RNAP pauses at AAA sequence. RNA terminator palindrome self-hybridizes, forming a stem-loop. this supercoil destabilizes RNAP-DNA binding, allows RNA to detach at A/T rich region.

Question 18

how does the Rho protein work?

Answer 18

Recognition site for Rho is the sequence of bases rich in C-A = rho utilization element. Rho moves rapidly towards transcription bubblee. it’s a helicase, unwinds DNA-RNA hybrid. RNA release, RNAP replease

Question 19

what does rifampicin do?

Answer 19

binds to beta subunit of bacterial RNAP near catalytic site. prevents RNAP from leaving promotor at start of RNA synthesis by blocking the channel on RNAP.

Question 20

what does actinomycin D do?

Answer 20

3 aromatic ring wit 2 short polypeptides, mimicks base pairs. intercalates between G-C base pairs, stops elongation by RNA Pol. Use in chemo

Question 21

what does death cap mushroom (amanitas) do?

Answer 21

inhibits eukaryotic RNA Pol II at low doses. synthesizes mRNA in eukaryotes.

Question 22

what is fragile X syndrome?

Answer 22

mutation of FMR gene. inherited menta lretardation. region b/w promoter and RNA encoding sequence expanded - insertion of 200 repeats of sequence CGG. inactivates expression of FMR 1 gene.