E4: DNA + RNA

DNA

3’ > 5’ phosphodiesterbonds join nucleotides

AGCT bases

has direction phosphate on 5’ and OH on 3’

carrier of genetic info (RNA in some viruses)

RNA

has direction

AUCG bases

base hydrolysis

only in RNA due to OH on C2

breaks down to nucleotides

acid hydrolysis

mild acid: removes purines from the polymer

strong acid: both DNA and RNA hydrolyze completely

exonucleases

enzymes that start at 1 end of DNA + remove nucleotides

5’ or 3’

can destroy DNA

endonucleases

cut bonds within the chain

restriction enzymes

a type of endonuclease

cuts at specific base sequences of DNA

rule of base equivalence

A=T

C=G (3 H bonds)

showed DNA was double stranded

Hydrogen bonding between bases

showed sugar-phosphate backbone was on the outside of DNA

good for aqueous environments

B DNA

right handed helix

axis of helix is perpendicular to bases

“normal”

Z DNA

left handed helix

mostly in regulatory regions

A DNA

right handed helix but axis is not perpendicular to bases

complementary DNA

DNA strands are not identical.. why [A] = [T] and [C] = [G]

supercoiled DNA

DNA forms a ring structure + keeps folding in

hyperchromic effect

DNA bases absorb more radiation at 260 nm if denatured > 2 DNA strands due to less interactions between bases

Tm

when ½ DNA is denatured

you need more heat to break more CG bonds

helps you analyze unknown DNA

CsCl gradient

used to analyze unknown DNA

heavy CG bases sink to the bottom

linear relationship between CsCl density and %CG

models of DNA replication

1. conservative

   • parent DNA stays in tact + new DNA is made

2. semiconservative

   • actual model

   • parent strands are separated new strand formed for each parent strand

3. dispersive

   • parent DNA is fragmented + pieced back tg

messelsen + stahl

grew DNA on radioactive 15N

showed DNA replication is semi-conservative

parental DNA: only 15N

1st gen: combination of 14N + 15N

2nd gen: ½ 14N + 15N and other ½ 14N

DNA dependent DNA polymerase I

studied by arthur Kornburg
looked at how DNA replicated

large protein, molar mass 110,000

needs a strand of Parental DNA, 4dNTP (N=ATCG), Primer (Small Piece of DNA or RNA)

replicase activity in 5 - 3 ' direction
3’ > 5’ exonuclease activity to remove nucleotide bases

removes primer it used DNA or RNA

told us that double stranded DNA is anti parallel bc

DNA repair mechanism enzymes

DNA polymerase I and II check for damage

1. endonuclease

   • makes single strand break where damage occured

2. exonuclease

   • removed damaged region of DNA

3. DNA dependent polymerase

   • resynthesizes DNA based on other strand

4. ligase

   • makes last phosphodiester bond

xeroderma pigmentosa

defect in repair enzymes in skin

prone to skin cancer bc cant repair damages

types of RNA

1. mRNA

2. tRNA

3. rRNA

messenger RNA

template for protein synthesis

all sizes

5’ capped with GTP to prevent it from being degraded

3’ poly A tail

introns cut out during processing > different proteins it codes for

transfer RNA

smallest RNA

brings AA in during protein synthesis

each tRNA binds specific AA (maintains genetic code)

has region that recognizes DNA template

ribosomal RNA

forms ribosome used to synthesize protein

3 sizes

23s: 100,000

16s: 50,000

5s: 40,000

5s at top of CsCl gradient and 23 at bottom

DNA dependent RNA polymerase

use DNA as template

only 1 strand of DNA is transcribed

500k molar mass

core enzyme

2 alpha, beta, beta prime subunits

transcribes + makes DNA randomly

holoenzyme

core enzyme + sigma factor

RNA looked like normal RNA

sigma factor

scans DNA for promotor region

promoter region

region of DNA that signals where to start transcription

upside of gene location

has palindromes

transcription steps

1. binding

   • RNA polymerase binds to DNA template

   • first nucleotides added (usually purines)

2. initiation

   • make 1st phosphodiester bond

   • RIFAMPICIN toxins inhibit this step

3. elongation

   • nucleotides added to make RNA

4. termination

   • RNA polymerase + RNA released

rho factor

termination factor

signals end of transcription

not associated w polymerase

ribosome

rRNA + proteins

needed for protein synthesis

eukaryotic: 80s = 40s + 60s

40s ribosome

rRNA + 30 proteins

60s ribosome

rRNA + 60 proteins

codons

3 nuclotides that code for AA

anti codon

3 nucleotides on tRNA that recognize codon

leucine tRNA synthetase

tRNAleu + leu must be both present or no rnx will occur

prevents disease

translation

using mRNA to make proteins

need right mRNA, all tRNA with AA attached + ribosome

aug

start codon

UAG

stop codon

stops translation

enhancer

binds molecules that stimulate transcription

operator

binds molecules that inhibit transcription

prion

infectious protiens that cause disease