mol bio special topics

crispr as a defense mech in bacteria

1. adaptation/ acquisition

   1. virus attacks bacterium

   2. saves small piece of DNA in protospacer

2. expression

   1. CRISPR gets transcribed

   2. processing —> gRNA w repeat + spacer

   3. each gRNA teams up w Cas

3. interference

   1. gRNA complex looks for matching seq

   2. if PAM is present (NGG —> foreign DNA) —> cut

   3. target viral DNA = protospeacer

   4. Cas protein cuts strands and bac nuclease destroys viral DNA

target any DNA seq w PAM

1. target DNA must have PAM (ex: NGG for Cas9)

2. design gRNA matching 20 bases before target seq

3. cas9 will cut 3 bases before PAM

PROCESS

after gRNA + Cas binding —> Cas9 makes ds break —» NHEJ creates mutations that lead to knockout

• allows mutagenesis in any species

base editing

changes single DNA wo cutting DNA

1. uses mutated cas9 that doesn’t cut

2. opens small, single stranded DNA bubble

3. sgRNA guides Cas9 to target

4. deaminase is fused to cas9 and changes chemical base on ssDNA

5. converts C—>U

6. becomes permanent bc U is treated like T so C—>G becomes T—> A

direct RNA targeting

cas13 mediated

cas13 + siRNA —> comparable knockdown efficiencies

imaging of DNA/RNA localization

dCas9 fused with EGFP can target specific DNA + mRNA to find loc in nucleus + cytoplasm respectively

ribosomes

non-covalent bonding (h-bonds) —> folded structure

• structures confer shape + f(n)

• tertiary active structure => folding of stem and loop

RNA regions confer catalytic properties

prokaryotic ribosomes

large and small sub only assoc when mRNA present

mRNA passes through tunnel in ribo

antibiotics bind to 16S rRNA of 30S small subunit at A site

why 16s binding occurs

small nucleotide diff in rRNA retain ribosome f(n) but create differential binding opportunities

• 16S = conserved region

doxycycline showed us what

has off target effects —> may lead to strange results

mitochondrial f(n)

tetracyclines —> mitonuc protein imbalance + mitochon dys

• in ppl => hearing loss + mito malf(n) in inner ear due to mito rib malf(n)

• in plants —> sick chloroplasts bc of chloro rib malf(n)

rRNA gene = phylogenetically similar to bacteria —> endosymbiosis

endosymbiosis

one bacteria engulfs another —> passed from gen to gen

evience of mito origination from bacteria

same size + shape

small, circular DNA

mito replication is autonomous from host cell

both have prokaryotic ribosomes

ETC is in cell membrane of bac and inner membrane of mitochondria

evolution of Eukaryotic cell

1. prokaryotes have infolding of plasma mem

2. cell develops nucelus + endomembrane

3. engulf prokaryotes (mitochondria)

why is rRNA a good phylogenetic marker

universally conserved

some highly conserved + var regions

length = good amt of material but small enough for sequencing

great way to map evolution

5 kingdoms were reclassified into

3 domains