BI216 exam 3

DNA structure

DNA structure

GC (triple bonds) stronger than AT (double H bonds)

antiparallel strands

major and minor groove

one turn of helix - 10bp

stem loop

pairing of ssDNA/RNA

4 bases needed for loop

Recombination

ds-breaks lead to recombination

meiosis - promotes genetic diversity (by Spoll protein)

mainly in DNA repair (Ds-breaks lethal if not repaired)hol

holiday juction resolution

via RuvC

branch migration

via RuvAB

gene conversion (definition)

number of genotypic outcomes is not equal as in starting genotype

gene conversion via mismatch repair/recombination

mismatch repair cant distinguish a correct strand → after recombination repair strand is equally as probable

each mismatch is resolved independently

mutation (definition)

heritable change in DNA sequence (DNA damage, mismatch/misrepair are not mutations)

Transition

no change in total number of base pairs

purine/pyrimidine → purine/pyrimidine (GC→AT)

Transversion

purine/pyrimidine → pyrimidine/purine (AT → TA, AT → GC)

missense mutation

wrong amino acid produced bc of base substitution

nonsense mutation

stop codon produced instead of amino acid

Indels

change in number of base pairs

insertion or deletion

in coding regions → frame shift mutation (change in reading frame during translation)

isolating mutants

selection - only mutants grow

screening - phenotypical differences between wild and mutant

mutagenesis

if repair doesnt occur

two rounds of replication for mutagenesis to occur

base excision repair

removes small lesiosns

1. DNA glycosylase cleaes altered base from sugar nucleotide → AP site

2. AP endonuclease makes a nick in backbone at AP site

3. DNA polymerase fills in gap by copying undamaged strand

4. DNA ligase seals nick in the backbone

nucleotide excision repair

removes large lesions

1. exposure to UV light leads to damage

2. thymine dimer forms

3. UvrB and C endonucleases nick strand containing dimer

4. damaged fragment is released from DNA

5. DNA polymerase fills in gap with new DNA

6. DNA ligase seals repaired strand

non homologous end joining

repairs DNA damage/ds-breaks formed during G1

1. catalytic subunit (KO70, KO80, DNA-PKCS)

2. nuclease cleans up damaged ends

3. DNA ligase rejoins ds-ends

frequent occurance of mutation (deletions or insertions)

direction of RNA synthesis

5 → 3

direction of DNA copying

3 → 5

sigma subunit

recognizes -35 sequence and -10 sequence

separates DNA strands from -12 to +2

sigma must rearrange for RNAP to leave promoter and continue elongation

termination of transcription (bacteria)

inversted repeat sequence

stem loop + multiple Us is a transcription termination signal

stem loop signals release of:

• DNA from RNA

• DNA from RNAP

• RNA from RNAP

need 4 bases for a loop

translation

mRNA to protein

tRNA mediate translation (each tRNA carries one particular amino acid)

tRNA

primary structure - tRNA sequence

secondary structure - clover leaf

start codon

AUG

stop codons

• UAA

• UAG

• UGA

open reading frame

open reading frame - no stop codon

6 total frames

mRNA sequences could be in one of three frames on non-transcribed strand

lactose operon

operon - coordinately controlled set of genes

LacZ

LacY

LacA

LacI

LacZ

encodes beta-galactosidase (b-gal)

LacY

encodes lactose permease (LacY)

allows lactose from outside of cell

LacI

encodes lactose repressor (LacR)

control protein of operon

binding sites in DNA for proteins

aka cis elements

LacO

LacP

LacO

lactose operator

binding site for LacR

LacP

lactose promoter

binding site for RNAP

LacR

active as tetramer

3 operators (binding sites for LacR)

• LacO1 at +11

• LacO2 +41

• LacO3 -82

repressing loop

multiple operators lead to formation of repressing loop

excludes RNA from binding lac promoter

lac control mutants

LacI-

LacOc

LacI-

mutant LacR cant bind to LacO

no repressor to stop transcription

LacZYA will always be on

LacOc

LacR cant bind mutant LacOc

LacZYA will always be on

glucose levels as regulators of Lac operon

LacZYA is on only when there is no glucose and only lactose is present

1. when glucose is low, cAMP is high

2. cAMP binds CRP proteins

3. CRP binds control region of lac operon and recruits RNAP

Lac operon control region

-10 consensus seq: TATAAT

-10 LacP: TATGTT

LacP mutagenesis

-10 sequence

TATGTT → TATATT (higher levels of mRNA)

TATGTT → TAGGTT (lower levels of mRNA)

assaying b-gal

cell extracts: ONPG (b-gal) → orange color can assay in spectrophotometer

plates: X-gal (b-gal) → blue color on plates

arabinose operon

when AraC bound operon is on

can bind to araO2, araI1, araI2

repressing loop forms in absence of arabinose

araBAD transcribed only in presence of arabinose and lack of glucose

restriction endonucleases

reorganize and cut spec seqs

purified from bacteria

EcoRI

restriction endonuclease

5’ overhang

5’-G|AATT|C sequence

KpnI

restriction endonuclease

3’ overhang

5’ G|GTAC|C

Rsa

restriction endonuclease

blunt end

5’-GT|AC (splits in the middle)

bacteria consensus sequence

TATAAT (-10 sequence)

eukaryotes consensus seq

TATAAAA (TATA box)

cloning (steps)

1. making recombinant DNA: cut vector with same restriction enzyme (EcoRI used)

2. mix digested vector and genomic DNAs together in presence of DNA ligase

3. creation of plasmid library

plasmid vectors

must have

• replication origin - plasmid can replicate as cell divides

• selectable marker - only cells that pick up plasmid will grow into a colony

• at least one unique restriction site - where fragments of interest are incorporated into vector

cDNA library

• collection of vectors, each with a unique DNA sequence derived from unique mRNA

• from mRNA (~2% of genome) - no introns, promoters, origins

cDNA library steps

1. isolate polyA+ mRNA from eukaryotic cell

2. add polydT primer (dATP, dCTP, dGTP, dTTP)

3. add reverse transcriptase → makes DNA copy of mRNA template

4. add Rnase (degrades mRNA)

5. DNA folds back on itself and seld primes

6. cut with restriction endonuclease

7. clone DNA fragment into vector

genomic DNA library

derived from genomic DNA (100% of DNA) - includes exons, introns, promoters, origins

base recognizing enzymes

GC - Arg409 (argenine)

TA - Gln45 (glutaminase)