Rifampin

inhibits during transcription

• G≡C  A=T
• hydrophobic interactions of bases (interior)
• Phosphodiester conections

DNA replication

Substrates

Products

Substrates: dNTPs

Product: Pyrophosphate

5-fluorouracil

(methotrexate)

• Replication inhibitor
• prevents formations of precursors (base, nucleotides purines, pyrimidines)

• Inhibit human topoisomerase II 
• treat neuroplastic diseases
• increases cutting by topoisomerase, or decreases resealing.

• in stem cells and cancer cells
• extends the 3' end of DNA
• use RNA templte AAUCCC as part of enzyme
• RNA primer synthesized by primase
• DNA polymerase adds the TTAGGG

• contributes to basal mutation rate
• T binds with G
• A binds with C

Missense

nonsense

sense

Missense: change in AA

nonsense: causes a stop codon to be formed

sense: no change in AA

• Fix spontaneous depurination/deamination
• remove nucleotides that lost their base

• Fix environmental damage, UV, smoking
• Fix T-T dymers
• T-T falls off, so nick, Ligase fixes nick

• Location identified by primary transcript
• begin GU end AG
• splicing done by spliceosome and SNURPS

UAG

UGA

UAA

• brings mRNA to ribosome
• requires hydrolysis of GTP
• initiates with AUG (MET)
• methyl guanosine cap binding complex binds to cap site on mRNA (rate limiting step)

glutamic acid residues

(requires vitamin K)

precoagulation factors and bone proteins

1. add manos-6-P to protein to acid hydrolase precursors

2. segregate protein from others in trans-golgi by binding to manos-6-P receptors at "clathrin pits"

3. clathrin coated vesicles bud off, bring protein to manos-6-P receptor

4. vessicle loses calthrin coat & fuses with endolysosomes.  at acidic PH protein separates from manos-6-P, Phosphatasein endolysosome removes P

Amplify DNA

1. DNA heated to break into SS DNA. 
2. Primers added, dna cooled SSDNA->DSDNA
3. Heat stable DNA polymerase and excess dNTPs added-> new complementry DNA strand made.

• genetic varients resulting from mutations causing a + or - of a restriction site.
• change in DNA frgment size after restriction digestion
• detectable by southern blotting
• RFLP analysis can be used if

1. RFLP closely associated with the disease-causing mutation
2. or mutation that gives rise to the disease is same as resulting in polymorphism.

good test for CF or Sickel Cell

• Specific to unique DNA sequence
• Used in PCR to find genotype
• Dot plot

• secondary structure of SS DNA is sequenze specific
• run in gell, if mutation, will migrate at a different rate because different secondary structure.

RNA half life.

• Short lived mRNA code for regulatory proteins like growth factor
• Long lived mRNA code for housekeeping

stability ↑ with 3' UTR stem loop.  & poly A tail.

• Produced using sequence complementry to selected mRNA
• bound mRNA sequence won't be translated in cytoplasm
• Drug Fomivirsen

Glycosaminoglycans (GASs) + Proteins

gell-like, resist strain, lots in cartalige/knee.

Fibrous protein

Collagen

• resistant to sheering force
• 3 helicle polypeptide α chains, each 3rd AA is a Glycine
• Synthesis requires Vitamin C
• deficient = osteogenesis, marfan

Occluding (tight junctions): form barriers

Anchoring (Desmosomes): connect cells

• Ca dependent cell-cell adhesion
• conects actin from cells together

• Cell surface carb binding proteins
• bind glycosylated ligands from 1 cell to another
• important on monocytes in bloodstream for rolling

• Ca independent cell-cell adhesion
• weak adhesion, more regulatory.

• Alpha and beta transmembrane chains
• Mediates cell binding to ECM
• Important for monocytes moving across endothelial cell.
• fibronectin = ligand

bind to platelet receptors

inhibit platelet adhesion and agregation

• G≡C  A=T
• hydrophobic interactions of bases (interior)
• Phosphodiester conections

DNA replication

Substrates

Products

Substrates: dNTPs

Product: Pyrophosphate

adds nucleotide to 3' end of growing strand

catalyzes formation of phosphodiester bond

• synthesizes short stranded RNA complementry to DNA template

Not in humans

• 3' to 5' exonuclease activity
• removes nucleotides not part of the double helix

Topoisomerase I

Topoisomerase II

I: catalyzes break in 1 strand of DS DNA to relieve tension.  new phosphodiester bond made.

II: catalyzes break in both strands of DS DNA to relieve tension.  new phosphodiester bond made

5-fluorouracil

(methotrexate)

• Replication inhibitor
• prevents formations of precursors (base, nucleotides purines, pyrimidines)

Anti Cancer drugs

(Doxorubicin)

• Effect template or priming ability of growing strand

treats UTI

inhibits DNA gyrase strand cutting reaction.

• Inhibit human topoisomerase II 
• treat neuroplastic diseases
• increases cutting by topoisomerase, or decreases resealing.

• in stem cells and cancer cells
• extends the 3' end of DNA
• use RNA templte AAUCCC as part of enzyme
• RNA primer synthesized by primase
• DNA polymerase adds the TTAGGG

• contributes to basal mutation rate
• T binds with G
• A binds with C

Base analog

who does bromouracil replace?

Alkylating agents

adds R group or OH

(eg) methyl bromide, or ethylene oxide.

Deaminating agent

A->

G->

C->

A-> Hypoxanthine

G-> Xanthine

C-> Uracil

Transitions

point mutation

Transition: pur-> pur.  pyr-> pyr

(common)

Point mutation

Transversion: pur->pyr or Pyr-> pur

Missense

nonsense

sense

Missense: change in AA

nonsense: causes a stop codon to be formed

sense: no change in AA

Sickel cell

thalassemia

sickle cell: missense glutamine -> valine

on beta globin gene

thalassemia: deficient in beta-globin or alpha-globin

fix errors from DNA polymerase

wrong nucleotide pairing

loss of mismatch repair gene = colon cancer

• Fix spontaneous depurination/deamination
• remove nucleotides that lost their base

• Fix environmental damage, UV, smoking
• Fix T-T dymers
• T-T falls off, so nick, Ligase fixes nick

• Damage from oxidation, chemotherapy
• BRCA1&2: homologous recombination.  mutation = breast cancer
• Fanconi Anemia: Mutation to DNA recombination repair enzymes

catalyzes synthesis of MRNA

inhibited by α-amanitidine

• needs RNA polymerase II & TFII
• TATA binding protein (TBP) binds to TATA to regulate

transcription factor plays a role in transcription &

DNA repair. 

if deficient = xeroderma pigmentosum (XP)

transcription results in RNA larger than MRNA. 

large RNA = hnRNA

introns cut out, donor and acceptor sites joined.

Capping

Polyadenylation

Capping: 5' end capped by a methyl guanosine residue, protects from degredation by 5' exonuclease

Polyadenylaion: 3' end high AAUAAA, enzme cleave 20 BP downstream.

• Location identified by primary transcript
• begin GU end AG
• splicing done by spliceosome and SNURPS

Thalassemias: heredetary anemia

point mutations at TATA or at intron-exon boundry.

inhibit bacteria DNA directed RNA synthesis

inhibits bacteria RNA polymerase.

inhibit enzyme stays bound to promoter

fights TB

AZT/DdI

inhibit reverse transcriptase

nucleotide analog

UAG

UGA

UAA

Aminoaceyl TRNA binds A site

peptidyl TRNA binds the P site

activity that catalizes peptide bond formation happens at large subunit.

• brings mRNA to ribosome
• requires hydrolysis of GTP
• initiates with AUG (MET)
• methyl guanosine cap binding complex binds to cap site on mRNA (rate limiting step)

eIF2

eIF4E

eIF2: inactivated by phosphorylation.  Heme inhibits phosphorylation

eIF4E: activated by insulin to start protein synthesis

1. Binding of aminoacyl tRNA to A site (GDP)

2. Peptide bond formation catalized by peptidyl transferase.

3. Translocation.  tRNA leaves.

stop codon

eRF" release factor

(GTP)

Lysosome

Proteosome

Lysosome: degrade extracellular proteins

proteosome: degrade intracellular (requires ubiquinone marker)

Bortezomid

(Velcade)

drug blocks proteosome activity

treat myeloma

adding of sugar to serine, threonine asparagine

not happen in bacteria

proline or lysine

in ER

requires ascorbate vitamin C

glutamic acid residues

(requires vitamin K)

precoagulation factors and bone proteins

1. add manos-6-P to protein to acid hydrolase precursors

2. segregate protein from others in trans-golgi by binding to manos-6-P receptors at "clathrin pits"

3. clathrin coated vesicles bud off, bring protein to manos-6-P receptor

4. vessicle loses calthrin coat & fuses with endolysosomes.  at acidic PH protein separates from manos-6-P, Phosphatasein endolysosome removes P

release regulated by glucagon, glucose, ARG

preproinsulin (has signal peptide)-> Proinsulin.  In golgi cleaved to make-> Insulin + C-Peptide

Insulin stored in cytosol in granules or vesicles untill release

1. Cut DNA using restriction endonuclease
2. Insertion into a vector (bacterial plasmid) to make recombinant DNA
3. Copy recombinant DNA in a cell based system (bacteria)

• Bacteria plasmid is cut with same restriction endonuclease as human
• Human DNA is inserted, sticky ends conect
• DNA ligase makes the conection.

plasmids produced from a digest of genomic DNA

contains entire genomic DNA

exons, introns, (everything)

Complementary DNA Libraries

(CDNA)

Plasmid from a digest of reverse-transcribed RNA

RNA->DNA

Contain DNA only from expressed genes

(Important stuff)

1. Only cDNA can be expresed in bacteria
2. coding sequence for cDNA joined to strong promoter in bacteria
3. 5'-UTR of transcript contains ribosomal binding sequence
4. vector has transcription & translation termination signals

Amplify DNA

1. DNA heated to break into SS DNA. 
2. Primers added, dna cooled SSDNA->DSDNA
3. Heat stable DNA polymerase and excess dNTPs added-> new complementry DNA strand made.

In situ hybridization

FISH

1. Probe added to denatured portion of DNA, and allowed to bind
2. Excess unbound probes removed
3. Site probe bound is identified.

FISH: uses probes of different colors to diagnose. 

Southern blotting

RFLP analysis

DNA fingerprinting

• DNA fingerprinting to detect DNA fragments containing specific sequences (eg) wild type of mutated gene.

1. Extract DNA from cell
2. restriction enzyme DNA-> DNA fragments
3. separation by size by electrofloresis
4. ID DNA by probe-hybridization

• genetic varients resulting from mutations causing a + or - of a restriction site.
• change in DNA frgment size after restriction digestion
• detectable by southern blotting
• RFLP analysis can be used it

1. RFLP closely associated with the disease-causing mutation
2. or mutation that gives rise to the disease is same as resulting in polymorphism.

good test for CF or Sickel Cell

• Specific to unique DNA sequence
• Used in PCR to find genotype
• Dot plot

• secondary structure of SS DNA is sequenze specific
• run in gell, if mutation, will migrate at a different rate because different secondary structure.

• When and how often a gene sequence is copied to RNA
• Activation: effect conversion of compact chromatin to extended structure
• Modulation: effect transcription factors

General: abundent, assemble on all genes transcribed by RNA polymerase II

Specific: few, bind to specific DNA nucleotide sequences.  bind as homodimer/ heterodimer

Processing of primary transcript

modifying synthesized molecules to make functional mRNA.

1. Add methyl guanosine cap at 5'
2. Add poly A tail at 3' for stability and translation efficiency
3. Removal of introns, exons conected by RNA splicing.

• DNA binding domain Identifies specific DNA
• Transcription activating domain accelerates assembly of general transcription factors at promotor site.

mRNA exits nucleus through pores

nucleases degrade mRNA before it can leave.

RNA half life.

• Short lived mRNA code for regulatory proteins like growth factor
• Long lived mRNA code for housekeeping

stability ↑ with 3' UTR stem loop.  & poly A tail.

regulate when mRNA is translated into protein

also initiation regulation

often proteins formed must be modified to be active.

like phosphorylation, gamma carboxylation

• Produced using sequence complementry to selected mRNA
• bound mRNA sequence won't be translated in cytoplasm
• Drug Fomivirsen

Glycosaminoglycans (GASs) + Proteins

GAGs: disachuride chans

(-), so repel.  H2o atracted.

gell-like, resist strain, lots in cartalige/knee.

protein with GAGs facing out (bottle brudh)

Hyaluronic acid (trunk of tree)

Fibrous protein

Collagen

• Protein fiber resistant to sheering force
• 3 helicle polypeptide α chains, each 3rd AA is a Glycine
• Synthesis requires Vitamin C
• deficient = osteogenesis, marfan

Fibrous protein

Elastin

• Rubber bands with knots
• small non polar AA residues, rich in Pro and Lys.  No hydroxyproline & hydroxylysine. 
• At ECM tropoelastin scaffolds to fibrillin
• Desmosine: 3 alysine residues + 1 lysine (knot)
• excess = Ehlers-Danlos (stretchy skin)

• Fibrolectin & Laminin
• has 3 integrins (binding domains) to collagen & proteoglycans.

holds 2 cells together

Occluding (tight junctions): form barriers

Anchoring (Desmosomes): connect cells

Communicating (gap): signal transfer

• Ca dependent cell-cell adhesion
• conects actin from cells together

• Cell surface carb binding proteins
• bind glycosylated ligands from 1 cell to another
• important on monocytes in bloodstream for rolling

• Ca independent cell-cell adhesion
• weak adhesion, more regulatory.

• Alpha and beta transmembrane chains
• Mediates cell binding to ECM
• Important for monocytes moving across endothelial cell.
• fibronectin = ligand

• Innermost layer of blood vessel (Intima)

1. Damage to endothelium
2. Monocite adheres to site and becomes lipid-ingesting macrophage.

TFP I: Protease inhibitor, inhibits VIIa and Xa

Anithrombin: inhibits Thrombin & Xa

destroys Va and VIIIa

1. Thrombin binds to thrombomodulin
2. Thrombin cleaves protein C to protein Ca
3. Protein Ca and cofactor protein S cleave & destroy F Va and F VIIIa

breakdown of fibrin clot

TPA released from endothelium binds to fibrin clots, activates plasminogen to plasmin

bind to platelet receptors

inhibit platelet adhesion and agregation

activates antithrombin

Heparin neutralized by Protamine