Topoisomerases

Topoisomerases regulate the supercoiling of DNA

reads 3' -> 5'

oh -> phos

Writes 5' -> 3'

Topoisomerase unwinds DNA Supercoil

Helicase – unwinds the two strands of DNA

•SSBs(single-stranded DNA binding protein) – keep the single strands apart during unwinding and copying

r

•DNA polymerase δ – synthesizes the complementary strand of DNA from 5’to 3’ leading strand

•Primase – RNA primer then DNA polymerase α & PCNA and then DNA polymerase δ

•RNaseH1 removes the RNA primers

•DNA ligase seals and completes the lagging strand

Lagging strand growth

strand lags after the fork

1kb piece

5->3

Ribosomesbind mRNA while mRNA is still being synthesized

 Multiple ribosomesbind to each mRNA, proteins are made rapidly

Longest peptides are on ribosomesfurthest from the start codonAUG

exonuclease cuts out damage

helicase melts double helix and damage is removed

DNA Polymerase I fills gap and DNA ligase seals strand

expression - low glucose, available lactose

lacY brings lactose into cell and lactose derepresses lac operon by preventing lacI inhibitor to bind to O site, low glucose means high cAMP binds to CAP to allow, RNA polymerase binds and translates, lactose converted to glucose by lacZ, 

Trp repressor activated by tryptophan and it repressing expressing when active

Concentration of tryptophan is:

high: doesn't need to make it, TrpR a repressor is activated preventing tryp from being made 3-4 loop RNA polymerase reaches loop and is stoped

Starvation: TrpR and TrpL deactivated, Tryp is made at max rate 2,3 loop stall ribosome, RNA polymerase makes Trp

reductive amination/transamination

draw

ketone-energy for the brain

1) CO2 + NH4 + 2ATP -> Carbamoyl Phosphate + ADP + H2O

2) Carbamoyl Phosphate + Ornithine -> Citrulline

3) Aspartate + Citrulline -> Argininosuccinate

4) Argininosuccinate -> Fumarate + Arginine

5) Arginine -> Urea + Ornithine

urea denatures protein

ammonium, ammonia, urea are toxic

2 ATP to dispose of urea

IMP to AMP

Inhibited by?

1) IMP + Aspartate + GTP -> Adenylosuccinate + GDP + P

2) Adenylosuccinate -> Fumarate + AMP

IMP = Inosine

AMP = Adenosine

*Inhibited by Accumulation of products

IMP to GMP

Inhibited by?

1) IMP + NAD+ + H2O -> Xanthosine + NADH + H+

2) Xanthosine + Gln + ATP + H2O -> Glu + ADP + PP + Guanosine

*Accumulation of product limits synthesis

1) Aspartate + Carbamoyl -> N-Carbamoyl-L-aspartate

2)N-Carbamoyl-L-aspartate -> Dihydroorotate + H2O

3)Dihydroorotate + NAD+ -> Orotate + NADH

4) Orotate + PRPP -> OMP

5) OMP -> UMP + CO2

or

1)UTP + Glutamine + ATP -> CTP + Glutamate + ADP+P

Hormone binds to receptor

alpha +GTP bind to adenalate cyclase to activate it = uses ATP for cAMP

GTP turns to GDP, deactives and aplha recombines with Y and Beta

cAMP increases activity of cAMP dependant protein kinases which in turn activate enzymes in glycogen breakdown and glucaneogensis (more glucose = more energy) and slow glycogen storage by making LESS F26BP

Hormone binds to PIP2 in cell membrane activating PLC

PLC catalyses hydrolysis of PIPP2 to IP3 and DAG

IP3 stimulates release of CA2+

DAG activates PKC

PKC phosphorylates CA2+ channel proteins controlling the flow of CA2+

[image]

Nucleotide

Nucleoside

Nucleotide = Base, Sugar, Phophoric Acid

Nucleoside = Base, Sugar

DNA/RNA Chain

Charge?

The top O- of the phophate replaces with the -OH on the 3' Carbon

C-O-P-OCH2

Negative charge due to phosphate backbone

A-T

G-C

Bonds?

A-T has 2 H bonds

G-C has 3 bonds

G-C is much harder to break

mRNA = messenger RNA = template for produce proteins

tRNA = transfer RNA = binds to target mRNA for protein production

rRNA = ribosomal RNA = translation of mRNA to polypeptide

Held together by disulfide bridges

Stimulates glucose uptake, glucokinase for G6P, Phosphofructokinase and Pryruvate dehydrogenase

activates glucogons syn and fatty acid syn

Diabetes

Type I

Type II

Reading direction

DNA Synthesis

RNA Repair

Proofreading

DNA Synthesis 3 - 5 adds new pair to 3

RNA Repair 3 -5 adds new pair to 3

Proofreading 5 - 3 adds new pair to 5

writes new strand oppostie

DNA Polymerase adds on nucleotides on the 3' end

ozaki fragments needed for lagging strand

primase starts ozaki and polymerase III completes it

exonuclease removes primer

Polymerase I fills primer gap

Ligase joins end

Prokaryotes: one origin of replication

Ozaki : 1-2 kb

Polymerase I and III

Eurk: Many origins of replication

Ozaki: .1 - .2kb

Telomeres to 3' of leading strand

rho independant

rho dependant

rho indenpendent: stem loops bump off RNA polymerase

rho dependant: stem loop causes RNA polymerase to pause, rho catches up and bumps off RNA polymerase

prokaryotic mRNA

Eukaryotic mRNA

Pro: mRNA code for several proteins in an operon

Euk: Code for introns and exons but exons are functional

Enzyme: DNA-dependent RNA polymerase but requires ATP, CTP, GTP, UTP, and MG2+

RNA Polymerase reads DNA 3-5 but grows 5-3 hydrolysis of pyrophosphate for energy

1) RNA polymerase has for subunits alpha, 2 Beta, sigma

sigma binds to promoter site T & A rich, and unwinds DNA double helix

2) sigma pops off and core enzyme continues and can make all three types of RNA

Polymerase I: rRNA

Poly II: mRNA

Poly III: tRNA

TFIID binds to TATA box follwed by Pol II and TF: A,B,F,H,E to begin transcription

DNA opens B,E,H comes of and Pol II moves along until termination site

Add a cap to 5' End and poly A tail to 3' in eurkaryotes

AG|G marks change point of exons and introns. introns are cut out exons bind together via spliceosome

Also cut for better loops in tRNA

Not normally expressed, Cataboloic, induced by substrate

lac operon

Normally expressed, Anabolic, Repressed by product

Trp operon

2 types of Protein Kinases

how do they recognize site

recognize site by adjacent proteins

inactive until needed unless cleaning

phosphorlyate proteins from .5 mins to 15 hrs

Serine/Threonine Protein Kinase

regulates metabolism

98% of protein phosphorylation

found through cell

Tyrosine Protein Kinase

regulates growth

.2%-2% of phosphate depend on cell division more when active

found throughout cell

Recognize Tert. structure

dephosphorylate phosphoserine/threonine

regulation of metabolism

5 types

found throughout the cell

Dephosphorylate phosphotyrosine

crtical in regulation of growth control

many many types

found throughout cell

Synthetase attaches amino acid to tRNA at the 2' or 3' Carbon but then is changed to the 3'

rRNA is 70s made up of a 30S section and a 50s Section

30s = 16s + 21 proteins

50s = 23S + 5s + 34 proteins

AA ->tRNA = 2ATP

translation =2ATP + 4GTP

1) 30S with IF1 and IF3 combine with tRNA+IF2+GTP forimng the 30s initiation complex on the start AUG codon a few steps down stream of the SD sequence in prok NO SD in Euk

2) IF1,2,3 leave with GDP and 50S joins 30s to make 70s initiation complex

3)Reads mRNA 5' -> 3'

EPA

5'-3'

A=incoming amino acid

P attachment

E=exit

4) EF-Tu brings AA-tRNA to A site hydrolizes and GTP and EF-Tu leave, leaving AA-tRNA

5)EF-G-GTP moves A site to P site hydrolyzes using GTP EFGGTP and GDP leave with A site open for next

6)RF1/2 recognized stop codon binds in A site transfer peptidyl group to H2O and release polyp chain, RF3-GTP uses GTP to remove RF1/2 and all leave

DNA is packaged so its 50,000x shorter, must remodel to gain access to DNA.

Methylation of Histones silence expression

Acetylation of histons by HAT turns on expression by reduce affinity of histones to DNA by neutralizing positive charge on AA

40S with 3 bind with eIF1 and eIF2-GTP-Met-tRNA to form preiniation complex

eIF4 bound to CAP on mRNA bind with pre to form iniation compex

Use ATP everything comes off leaving just 40S, 60s joibs and translation starts

Fe in Heme group activates HCR

HCr uses ATP->ADP to make eIF2->eIF2-P & eIF2B-P

Endocrine

Paracrine

Autocrine

Endo: Target cells are distant from hormone making cell

Para: Target cells are near hormone making cell

auto: hormone maker is targer cell

Initiation
–Ribosome-binding site (RBS codon) on mRNA allows binding to 30s subunit
–Formylmethionine tRNA binds AUG (start) codon the Formyl group will be removed later
–50s subunit (shell) docks to 30s subunit –fMet-tRNA occupies the “P”site

•Elongation
–Next charged tRNAbinds to “A”site
–Peptide bond forms

•Translocation
–Ribosome moves from one codonto the next and the empty tRNAis released from the “E”site
–Next charged tRNAbinds to empty “A”site

•Process continues until stop codon

trna binds to codon -> ribosome binds at trna to p site and is added to chain -> trna binds to codon at a site-> ribosome move so p->e and a->p -> E codon ejected and new trna binds a site.  read p site to chain