Cell generates its own signal

(ie it secretes signal that is boudn by receptors on itself)

•  common in cancer cells - cells generate 'divide signal when it shouldn't, binds to that signal and as a result, divides when it shouldnt

hormone binding is often rapidly relatyed via multipe steps to ultiple proteins in a 'cascade'

Cascade - permits for rapid amplification of signal and more control by having more steps that can be regulated.

• Many signal transduction pathways involve kinases and g-proteins
• Kinsases phosphorylate substrate chaning activity of substrate, differents kinds of kinases phosphorylate different amino acids.
• Most common kinases are S/T
• S = Serine = Ser
• T = Thronin - Thr

Signal (or at least initial) is often a hormone.  The hormone binds to one or more receptors to initiate signal transduction process.

• Typically upon receptor hormone binding signla is related through cell via many different steps.

methyl group attached to histones.  generally increases packing and thus generally reduces transcription.

• Can suppress acetylation
• HP1 (heterchromatin protein 1) can bind to methylated histones and promote attachment of the chromatine containing the methylated histones to the nuclear lamina.
• This puts chromatin at periphery and promotes formation of heterochromatin.
• HDA3 (localized at lamina) - deacetylates the histone, further increasing the packing.

• Houses DNA thus site of DNA copy and transcription
• Central Dogma
• DNA replication then RNA transcription then Protein Translation.
• example of compartmentalization
• Compartments are structures in the cells that can segregate their contents from the rest of the cell. Compartments -
• Protect - keep materials sequestered from other materials
• Organize - keep similar things together, concentrate materials, what you want where, this makes faster reactions.
• Additional scope for regulation

Modulation of Histones _ DNA interactions

Histone Acetylation

Histone Acetylation - attachment of acetyl to histones

• weakens histones DNA binding
• Associated with reduced DNA packing
• Can increase transfer lion
• Can be revered by histone deacetylases
• Histone acetylation - attachment of acetyl group to histones - done by proteins with histone acetyle transferase (HAT) activity.

Core histones  = H2A, H2B, H3, H4

• At most basic level controlled by histones.
• Two copies of each to make octomer
• Octamer serves as strucutre to hich DNA wraps
• DNA + octamer = nucleosomes (beads on a string)
• Naked DNA = 2nm, beads on a string = 10nm width

Histone H1 - Next level of packing - gathers core histones - nucleosomes and forms (30nm fiber) solenoid.

• Higher levels of packing exist
• Chromosome width = 700nm

~ 3 x 10^9 base pairs

~ 25K genes that make up 5 % of genome

The rest is extra - some is responsible for regulation

Region of repetitive DNA to which microtubules attach during spindle assembly in mitosis

DNA and proteins that bind to DNA

Euchromatin -  center of nucleus, packed loosely, site of genes being actively transcribed.

Heterochromatin - packed tightly, typically near nuclear periphery, site of genes that are not being actively transcribed.

General observation - There is an inverse correlation btw the extent to which DNA in chromatin is packed adn the degree to which it is transcribed.

Attached to the INM, meshwork of lamins

• Lamins A, B, & C - form strong, linear structures.
• Lamins attach to INM by:

1. lamin phenylation (attached to a lipid group), lipid is hydrophobic and inserts into INM
2. Binding of IMPs to INM - Lamins bind to LBR, LAP, Sun
3. Lamins bind to Nup 153 ( in pore)

• Functions: Mechanical strength, may regulate gene expression.
• Diseases: progeria - premature aging, muscle dystrophies.

• very different from ONM in composition (lipid and protein)
• Harbors serveral IMPS (ex. LBR - lamin binding receptor, LAP - lamin associated protein)
• Sun proteins - IMP of INM bind to KASH in lumen of N.E. to form bridge from cytoplasm to nucleoplasm.
• INM - defies nucleoplasmic boundary of NE, also helps anchor protein in cell, binds nuclear lamina.

• Continuous with the Rough Endoreticulum
• has several integral membrane proteins (IMPs - proteins that pass through the membrane, ex. KASH protein on ONM)

• Has potential to move through cell membrane
• Receptors usually found inside the cell
• Receptors are usually transcription activators
• Response usually include change in transcription (also possible changes in metabolism or developmental fate).
• Lipid soluble hormone signaling is typically endocrine (best known example, steroids)

Origin replication complex - made of six proteins (Orc 1-6)

• acts as landmark - binds to ori for most of cell cycle and acts to identify oris as places to start replication.

Copying of DNA  - a semiconservation process

• DNA strands seperated, act as template for new strand
• Post replication two pieces of DNA both ahve one new and one old strand.

Preparation for replication

• Results from the assembly of Pre-RC (pre-replication complex ) at Oris (origin of replication initiation)
• Licensing is restricted to M-Phase and beginning of G1.
• Inhibited by CdK and Ddk.

• Actual process of Replication, restricted to S-Phase.
• Requires High activity of Cdk and Ddk (this is opposite regulatory switch of licensing).

• Begins at Oris (origins of replication initiation)
• Replication bubble forms, forks move away from bubble expanding DNA bubble,
• When bubbles merge, replication is complete
• Replication is extremely accurate due to accuracy of DNA polymerase.
• Cells copy genone only once per cycle and completely copy it.

Licensing

Firing

- this ensures copying only happens once.

• cytoplasmic IFs provide cells w/ mechanical strength
• help prevent cells from rupturing in response to physical trauma.
• Different IFs for different cells
• High tensile strength
• Lots of force to break
• Stronger than actin filaments
• comprised of IF proteins
• more varied in structure than actin and tubulin
• much less dynamic
• don't bind to nucleotides
• very stable when self assembled
• Non-polar
• no-associated motors

• Bind to and hydrolyze GTP
• When bound to GTP they are active, when bound to GDP they are inactive. 
• used as switches to control signal transduction pathways.
• When pure they are very poor enzymes (hydrolize nucleotides very slowly)

GEFs - Guanine (nucleotide ) exchange factors - stimulate exchanged GDP for GTP by G- proteins thus activating them.
GAPs- GTPase activating proteins - promotes GTP hydrolysis by G-proteins thereby inactivating them.

Move chromosomes during mitosis (critical Function) - requires motor proteins and MT assembly/disassembly

• In non-dividing cells, MTs direct trafficking of different cell components
• Dependent on motor proteins
• MTs can move whole cells with cilia and flagella
• Regulate signaling pathways.

Microtubule organizing center

• cell structure where MTs are nucleated and from which they extend to cell periphery.
• In most animal cells the centrosome acts as the MOTC - this is made of two centrioles
• Centrioles are surround by periocentriole material rich in gamme-TURCs.

Use energy provided by ATP to walk along microtubules.

Kinesins - move towards plus (+) end of MTs

Dyneins move towards minus (-) end of MTs

• These transport things in the cell and can also selectively disassemble MTs from one end to the other.

Regulated - transcribed only in response to certain extracellular cues.  Only in certain types or maybe only certain times, ex. keratin 14

• typically have closed promoter - core promoter is associated with nucleosome. Nucleosome keep GTF and Pol II from having easy access to the core promoter.
• Can be turned on by the combined action of upstream regulatory elements and so called activators.  When cell is in need, activators are recruited to upstream regulatory elements and turn on gene.

Pol II transcribes region and generates immature transcript.

Termination of transcription occurs @ some point beyond polyadenylation signal.

TF II D - 1st GTF to bind core promoter, binds to TATA box.

TF II H - has kinase activity, phosphorylates pol II thereby activating pol II, also has helicase activity and seperates (unwinds) DNA so pol II can access template strand of gene.

Pol II - large complex protein with many subunits, in many genes, recruitment of Pol II to core promoter is rate limiting step.

Process - TF II D then TFIIH then Pol II then TFIIE then TFII A, B, F.

General transcription factors (GTFs) - needed most or nearly all mRNA transcription - TF II - transcription factor II (letter)

TF II (A,B,C,D,E,F,H) bind in particular order to core promoter.

Roles of GTFs include:

1. Bind to core promoter and target Pol II to core promoter.
2. Position Pol II so it can start transcribing at +1
3. Activate Pol II - Promote transcription.

work liek adapators - have anticodon at one end (can hybridize to specific codon) and at other end they have an amino acid.

• Different tRNAs carry different amino acids and can hybridize different codons.

Refers to mechanisms by which cells receive process and respond to information

• important because it is -needed for responses to internal and external changes and challenges faced by the cells - Abnormal and inappropriate signal transduction.

Responses includes: Transduction, cell division, cell viability, cell developmental fate, cell morphology.

comprised of exons and introns

• different genes have different numbers
• different exons and introns may be transcribed by the same gene.
• exons end up in final transcript
• introns are removed during processing
• transcribed region (exons) are represented in protein
• Poly A signal - 6 nucleotides directs polyadenylation during transcription.

S= Serine - Ser

T = Thronine = Thr

thousands of proteins at any given moment are phosphorylated on S or T.

Tyrosine (aka Y or tyr) kinases - much much more rare, at any given moment very few proteins are phosphorylated on Y

Dual specificity kinases - can phosphorylate substrates on S, T, Y - are very rare - most are induced during cell division signal transduction.

many more cycles of DNAPA binding priming etc. than leading strand. 

• More DNAPA on laggin strand and more DNAPE on leading strand.

Poliferating cell nuclear antigen - aka gliding clamp - wraps around single stranded DNA, binds DNA polymerase D or E to it and keeps them attached to single stranded DNA.

Read template Strand 3' to 5' and polymerize 5' to 3' - need free 3

 end to start polymerizing.  Do not have primase activity, have proofreading activity, lets them remove RNA from DNA and fix errors or mismatches.

Katanin, Spastin

• bind to MT lattice and remove tubulin dimer (suses ATP as energy source)
• removal of dimers break M.T.
• This breakage results in disassembly because it exposes GDP tubulin

~50- 200 nucleotides

• core promoter ~ 50 nts, positioned adjacent to transcribed region, contain TAT box, ~7-9 nts often w/ TATA sequence.  Acts as binding site for general transcription factors and Pol II.

Can positively or neg. regulate access of transcriptional machinery to core promoter.

+1 position of first nucleotide that will be represented.

Origins of replication initiation -

vary according to organism - 50-1000s base pairs.

ex. MAP - 1 (microtubule associated protein -1), MAP -2 -

Stabalize microtubules, supress catastrophe, some bundle microtubules.

Pol I - transcribes large rRNA

Pol II - mRNA, miRNA, some snRNA

Pol III - tRNA, small rRNA, snRNAs not by Pol II

• Read template 3' to 5' polymerize 5' to 3' do not require free 3' end.  Do not have proofreading activity.
• Following or during transcription, transcripts can be modified.

generation of RNA transcript using DNA as a template

• similar to replication, has info needed for transcript formation.
• occurs through most of cell cycle (early M, G1, S, G2)
• as chromosomes get tightly packed during M, transcriptional machinery cannot access genes.
• Only a fraction of the genome is transcribed - genes can be transcribed 100 to 1000 of times.
• Compared to replication, transcription is inaccurate - DNA is forever, mRNA comes and goes.

can recruit co-activators and nucleosomes remodeling complex to promoter

Co-activators - Have 1) HAT activity - weakens histone interaction can bind and recruit GTFs and Pol II to promoter

Nucleasome remodeling complex - can reposition or completely remove them from the DNA

The coactivators and nucleosome remodeling compleses thsu collaborate to remove/displace nucleosomes from core promoter and to provide local source of transcription machinery.

Generation of protein using mRNA as a template

• Takes place in the cytoplasm and not nucleus.
• Occurs throughout the cell cycle, even during M-Phase
• A given mRNA may be translated hundreds of times in a cell cycle.
• mRNA template is read 5' to 3'
• Protein is made in the N-amino to C-carboxyl terminal direction.
• 3:1 correspondence btw nts in template and amino acids in polymerizing protein.

very short range signal source is right next to target cell, very common in development.

Signal is bound to the cell that makes it so signal cannot move away.

Commonly used during growth factor signaling

ie EGF, NGF (nerve), and PDGF (Platelet derived)

-commonly paracrine

• Response is often increase in cell division

General Scheme: initial signal is growth factor which binds to receptor that spans the plasma membrane.  Binding occurs outside the cell and results in an increase in tyrosine kinase activity(by the receptor) inside the cell.

S/T (serine/Thronine) kinase - when active it phosphorylates S218 and S222.

These phosphorylations activate mek

Serum Response Factor - SRF is a transcription factor when active, SRF bind to upstream regulatory elements of several genes and promotes their transcirption target genes and promotes their transcription

Target gene includes those that encode for Fos and Jun.

1. Eventually EGF secretion at would no longer occurs
2. Cells have phosphotases - protein that dephosphorylate proteins (ie remove phosphates from their substrates).  This counteracts kinases in EGF pathway.
3. Internalization of EGFR and EGF complex, cell bring EGFR-EGF via endocytosis then send it to the lysosome, here is it degraded.

Ras-GAP - promotes Ras inactivation by promoting GTP hydrolysis.

Singaling by corticosterone (aka cort)

• Is often considered a stress hormone
• in response to steress cort is released and promotes responses that help organism cope.
• One response controlled by cort is pepsinogen secretion into stomach
• Once in stomach pepsinogen is converted to pepsin which is used for protein digestion
• Thus orginism has greater long term energy mobilization.

Adrenal gland - synthesize and secrete cort into blood

Cort - Corticosterone - a lipid soluble hormone synthesized from cholesterol

Chief cells in the stomach lining.  Chief cells respond to cort by secreting pepsinogen into stomach then express cort receptors

Transcription activator - typical example of lipid soluble hormone receptors - has a tripartite strucuter - protein has 3 functional modules.

TAD -Transcription Activating Domain- Up regulates transcription of target genes by binding to and recruiting coactivator and nucleosome remodeling complexes.

DBD - DNA Binding Domain - responsible for putting receptor on proper target genes by binding to upstream regulatory elements of cort. target genes.

HBD - Hormone binding domain - binds to cort and is required to get receptor into nucleus after cort binds.

Hormone Response Element - example of upstream reg. element of cort

HRE = 5'-AGAACAATTTGTTCT-3'

Possibly most common form of signal transduction, typically paracrine or endocrine

• Often extremely fast due to signal amplification.
• Hormones are water soluble receptors and usually 7-pass PM IMPs
• When bound to hormone, receptors signal by acting as GEFs for heterotrimeric G-proteins in cell.
• Heterotrimeric G-proteins have 3 subunits - connection btw receptor and g-protein much more direct (relative to receptor tyrosine kinase signaling).
• Many different responses can be triggered by g-protein coupled receptors include - change in transcription, metabolism, or developmental fate.
• Ex. Adrenaline Signaling

Occurs in response to very acute stresses, by promoting generation of readibly useable energy source - ultimately ATP, immediate output of pathway is production of glucose (which is then used for ATP).

Synthesize and secrete adrenaline. 

Adrenaline - water soluble, synthesized from tyrosine.

made of alpha and beta and gamma subunits, just know alpha.

Gs-Alpha - When active (GTP bound) bind to and activates adenylate cyclase.

Binds to and activates PKA

A cyclic nucleotide is able to diffuse extremely quickly through the cell.  It is considered a second messeger.

2nd messengers - are small non-protein signaling molecules or ions that have in common the feature of high mobility (1st messenger = hormone)

A-receptor to Gs-alpha 10

AC to cAMP 10

PKA to PK 10

PK to GP 10

GP to glucose 10

potential 10^8

1. Adrenaline will not be secreted forever
2. Phosphotases counter act activity of kinases
3. Adenylate cylcase promotes GTP hydrolysis by Gs Alpha (acts as GAP)
4. cAMP is converted to AMP by phosphodiaterases.

EB1, Clasp

Bind to plus end of M.T.