Lect 2 in class

Question 1

Why does phenyl extraction work?

Answer 1

All DNA nucleotides will be negatively charged at neutral pH because the base and sugar will have no charge and the phosphate will have a negative charge.
This means it will be hydrophillic.
Protein is usually less hydrophillic

Question 2

Why does DEAE chromotragraphy work?

Answer 2

because the DNA is -1 charge at neutral pH, DEAE is positive charge, if it is the stationary phase it will attract DNA.

Question 3

Why does electrophoresis work?

Answer 3

Buffers are in 6.5 - 7 range, so that all DNA have -1 charge per nucleotide. All molecules equally pulled. The nature of the agarose matrix is that it is easier to move through if you are smaller, as you can fit through smaller holes. So smaller DNA moves faster.

Question 4

N3 is minor or major groove?

N7 is minor or major groove?

Answer 4

Minor

Major

Question 5

Given that N7 is the dimethyl sulfate for guanine vs N3 for adenine, which will dimethyl sulfate bind faster to?

Answer 5

Guanine, its site is in the major groove and is more accessible (10x more accessible)

Question 6

Given that N7 is the dimethyl sulfate for guanine vs N3 for adenine, which will dimethyl sulfate bind faster to?

Answer 6

Guanine, its site is in the major groove and is more accessible (10x more accessible)

Question 7

What does nitrous acid do to DNA?

Answer 7

Deaminates N4 of dC to ketone which makes it dU

Question 8

What does nitrous acid do to DNA?
Predominately targets what?
Why is this used?

Answer 8

Deaminates N4 of dC to ketone which makes it dU
ssDNA
Provides fairly random mutations

Question 9

Hydrazine:

what happens at 5M NaCl conc. ?

Answer 9

removes C and T

becomes specific for C removal

Question 10

Hydroxylamine:

Answer 10

Modifies C to a form which can bind to A

Question 11

Acid and base reactions know completely.

The nucleic acid base reactions just know the outcomes.

Answer 11

`

Question 12

Bisulfite

Answer 12

Works best on ssDNA, deaminates dC to dU

Question 13

All initial ribozymes found had what structure?

Answer 13

RNA with secondary structure which bound divalent cations (magnesium), which helped for nucleophilic reactions to occur

Question 14

Write how the numbering occurs on around circle of the hammerhead.

Answer 14

Check lect1 slide 32 to confirm

- I believe the hammerhead cleaves itself

Question 15

What does the hammerhead ribozyme do?

Answer 15

It cleaves one nucleic acid.

Question 16

What does the hammerhead ribozyme do?

Answer 16

It cleaves one nucleic acid.

Question 17

How does the hairpin rybozyme and the hammerhead ribozyme differ?

Answer 17

The upper strand of the hairpin ribozyme can be provided in trans. So it can use sequence

Question 18

Group 1 intron splicing:

Answer 18

Free guanine is activated by a metal cation attacks, to attack phosphate, taking it with it. The now open 3’ OH is activated by metal cation, attacks the 5’phosphate of the other intron cleavage site.

Question 19

Group 1 splicing (intron splicing?):

Answer 19

Free guanine is activated by a metal cation attacks, to attack phosphate, taking it with it. The now open 3’ OH is activated by metal cation, attacks the 5’phosphate of the other intron cleavage site.

so first releases 5’ intron, adding a G

Question 20

Group 2 splicing:

Answer 20

Same mechanism as Group 1 splicing. However instead of free G nucleoside, we have a CIS cleavage using a branched RNA secondary structure

Question 21

If you get a lariat structure, and treat it with base, what are the products you get?

Answer 21

Know where your phosphates are going, what ends up a hydroxyl

Question 22

Group 2 introns encode:

Answer 22

maturase, endonuclease and reverse transcriptase

Question 23

The only important thing about the hepititis delta virus ribozyme:

Answer 23

Both sides of the encode a ribozyme (i think)

Question 24

Droshe:

Answer 24

Cuts the hairpin structure out of pri-microRNA

Question 25

Exportin 5:

Answer 25

escorts miRNA out of the nuclease

Question 26

DICER:

Answer 26

Cleaves hairpin into ~ 22bps with 3’ overhangs. bound tightly. Need helicase to unbind strands.

Question 27

RISC:

Answer 27

Binds to

Question 28

Northern hybridization

Answer 28

Look it up hun

Question 29

CRISPR

Answer 29

Uses RNAs to degrade foreign DNAs

Question 30

CAS identifies foreign DNA (non-chromosomal DNA), clips pieces of it, inserts it into the genome of the CAS system between repeat spacers. So we have stored some of the DNA.

Answer 30

Next time the virus comes… Spacers are transcribed made. They are cut into each of their units.
It also has a motiff which double checks that the DNA if indeed foreign

Question 31

Explain G1 intron splicing.

Answer 31

A free GTP attacks with its 3’ OH a phosphate. It displaces the 5’ end, and leaves a GTP at the 5’ end of the strain. The 5’ end which was displaced attacks usually a uracil more 3’ end. This cuts the intron out of the strand (intron now has a G at its 5’ end.

Question 32

Explain G2 intron splicing.

Answer 32

An adenine, more 3’ on the molecule, attacks a more 5’ OH with its 2’ OH, cutting it. The 5’ strands 3’ OH attacks downstream of the lariat, and cuts the lariat off connecting the exons.

Question 33

Maturase:

Answer 33

A protein which splices together RNA or DNA