Lecture 17

Question 1

All DNA pol….

Answer 1

have essentially the same structure and proof reading activity

Question 2

What is specific to pol 1?

Answer 2

5-3 exonuclease activity

Question 3

What happens if dna pol makes a mistake?

Answer 3

Has to unwind, which is really unlikely to occur if there is no mistake.

Question 4

Nucleotide excision repair.

Answer 4

System recognizes helix (backbone) distortions, not specific chemical groups or adducts
Repairs UV photoproducts, among other lesions
More common than DNA photolyase
thymine dimers make things bulge out, this will be recognized
UvrABC endonuclease= ABC scinuclease
-cuts on both sides of the damage
UvrD
-helicase: unwinds to release
Pol I, DNA ligase
-replace and fix

Question 5

Xeroderma pigmentosum

Answer 5

genetic diseases of excision repair
in humans NER utilizes 16 different proteins. Mutations in many of these Cause XP
Convergent evolution; actual proteins are all different, same process

Question 6

DNA mismatch repair

Answer 6

(in e coli; humans lack MutH)
Specifically for errors made DURING replication

third level: look to see if there was damage

must happen soon after replication so you can determine which strand has mismatch

Must recognizes the mismatch
MutL and MutS act like a motor together (like atp synth)
Utilize ATP

Endonuclease- cuts DNA
bacteria methylate GATC, this is the new strand- know that the opposite sequence is the strand b/c doesn’t have the methylation (hemimethylation)
eukaryotes have the same process but no MutH protein and no methylation
We don’t know how we recognize which strand is new
Hereditary nonpolyposis colorectal cancer syndrome- mutations present in 1 in 200 in the US population
Really specific disease- that’s weird. Mutation in Muts and MutL?

Question 7

Chemical Mutagens: alkylating agents

Answer 7

will often cause misfiring
alkylating agents include:
-nitrogen mustard (also chemotherapeutic, targets rapidly dividing cells)
-ethylnitrosourea
-MNNG
second two: mostly used for creating mutations in labs
a common alkylation product is O6-methyl-guanine
base pairs with either T or C
This and other O6-alkylguanine adducts can be removed by MGMT, one shot direct repair protein that transfers the alkyl moiety to an active site Cys residue
a lot of energy just to fix one small thing
NOT an enzyme! the protein can only be used once, then is degraded.

Question 8

Chemical mutagens: oxidizeres

Answer 8

over 100 different oxidative DNA modifications
-deamination of cytosine to uracil (which pairs with adenine)
-adenine to hypoxanthine, which pairs with cytosine
Other powerful oxidizers are produced through normal metabolism- not just diet

Question 9

Base excision repair

Answer 9

1) The defective or incorrect base is removed by a DNA glycosylase. Several different DNA glycosylases recognize different problem bases. Leaves backbone and OH
2) The backbone is cleaved at the AP site by AP endonuclease
3) DNA polymerase removes the naked sugar/phosphate and fills the gap which is then sealed by DNA ligase
Cut out AP site (sugar phosphate bond) and just leave a nick which DNA ligase would fill in after synth of one nucleotide.
Lose your base- still a backbone, just no base
ap= abasic (apurinic or apyrimidinic)
note that this mechanism provides a rationale for why dT not dU is found in DNA.
detect mismatches or incorrect structures in DNA. Recognize weird/wrong bases.

Question 10

Uracil-DNA glycosylase catalyzes base excision

Answer 10

1) This structure shows UDG bound to a DNA duplex containing a dU: dG mismatch
2) It shows the state of the enzyme:substrate complex immediately after hydrolysis of the of the glycosidic bond
3) note the base-flipping mechanism of the enzyme
4) note distorted DNA backbone
* also known as a DNA glycosidase

Question 11

why an RNA intermediate?

Answer 11

Regulation… and maybe history

Question 12

To do different jobs, differentiated cells must make different proteins in different places

Answer 12

rna enables us to do regulation
harder to regulate with DNA
other reason: RNA probably came before DNA

Question 13

Why an RNA intermediate

Answer 13

hydroxyl group on 2’
makes rna less stable b/c can attack phosphate group nearby which leads to spontaneous breakage
also can do a lot of chemistry that DNA can’t do- can be catalytic

Question 14

Why RNA int?

Answer 14

history probably- RNA world hypothesis
Probiotic world: condensation of sugars, bases, phosphate, and random polynucleotides
RNA world: rna genomes, rna enzymes
RNP world: protein synthesis, protein enzymes
DNA world: dna genomes

Question 15

Biggest issue in regulation:

Answer 15

where and when to turn on

also, amplification

Question 16

why an rna intermediate?

Answer 16

amplification
mrna molecules are messages sent to the factory
can make as many of them as you need in the factory (ribosomes)

Question 17

rna polymerization

Answer 17

same chemistry as DNA replication
RNA- only using one strand (template/antisense) to synthesize
always going 5-3
still have to unwind
LOOK AT DIAGRAM
dna coding strand= sense strand
dna tempalte strand = anti sense strand (this strand base pairs with the nascent RNA)
RNAP= RNA pol
bigger/more complicated than dna pol
dna used rna b/c rna does not need a primer- remember that rna pols can initiate without a primer!

Question 18

RNAP catalytic subunit architecture is evolutionarily conserved

Answer 18

convergent evolution
2 mg2+ in active site pyrophosphate as leaving group and allow attack
structure: more like crab claw than hand

Question 19

Replication vs transcription

Answer 19

Both go 5’-3’
replication uses dNTPs and transcription uses rNTPs
replication needs a primer; transcription doe snoop
replication goes at 1000 nt/s and transcription goes at 50
rna way smaller (just one gene) so it doesn’t need to be as fast
replication has sliding clamp, transcription doesn’t really need one, rna pol acts as sliding clamp
error rate is 10^-10 vs 10^-4
don’t care as much with transcription b/c not going to be inherited

Question 20

what are promoters/what do they do

Answer 20

cis-acting dna sequences called promoters tell the transcription machinery where to initiate
most important: when to initiate. most regulation happens at initiation
prok:
activators bind and stimulate transcription
repressors bind and inhibit transcription
highly conserved with these sequences (in prok or euk or both?)
euk: dna seq upstream of the gene (Further on 5’ on the sense strand)
enhancer
(second, first)
CAAT box or GC-rich regions
tata box
cap site
+1

Question 21

Three eukaryotic DNA dependent RNA polymerases

Answer 21

Prok- 1 rna polymerase
euk: multiple
RNAP 1 makes rRNA, makes ribosome
RNAP II: makes mRNA, makes protein, protein goes to ribosome
RNAP III: makes tRNA

Question 22

RNAP II and the GTFs together form the pre-initiation complex (PIC)

Answer 22

euk: on its own, can’t recognize a promoter
need general transcription factors
TBP = tata binding protein
help rna pol recognize a promoter

Question 23

PIC: Pre-initiation complex: general transcription factors (GTFs) & RNAP II assemble at a euk promoter. What is the TBP important for?

Answer 23

TATA binding protein (TBP) is a key subunit of TFIID
TBP introduces a 45 degree bend into the double helix, locally untwisting the DNA
TBP is a universal GTF, required for RNAP I, II, and III initiation

Question 24

What do enhancer sequences do?

Answer 24

regulate the activity of core promoters
Specific enhancer sequences bind different activator transcription factors
each gene has a different array of enhancer sequences

Question 25

What is a mediator?

Answer 25

many activators regulate transcription with a mediator, a huge protein complex of greater than 20 subunits
required for efficient transcription from ALL RNAP II promoters
mediator complex sits between activator protein and RNAP II

Question 26

Heterochromatin and euchromatin

Answer 26

Regions of repressed and activated transcription
heterochromatin is condensed- basically off
euchromatin: on?? dispersed or extended