371 terms

Biochem 4

Chromatin structure, and charge on DNA/Histones
Chromatin: DNA + histone proteins wrapped in a tightly compacted situation
-DNA is negative; histones are positive
Mitochondrial genome has ... (3)
-circular molecules
-multiple different copies
-encodes 13 proteins
Euchromatin vs Heterochromatin
Euchromatin: light stain, loose DNA, allows xC

Heterochromatin: tight DNA, dark stain
Active Transport into nucleus
Importin binds molecule
-taken into nucleus, importin comes off
-importin binds ranGTP, taken back into cytosol
-Ran-GAP cleaves complex; importin stays in cytosol
Active Transport out of nucleus
Exportin binds mRNA; binds RanGTP as well
-complex exported to cytoplasm
-Ran-GAP cleaves complex
on 3' end of DNA; prevents digestion by enzymes

-Telomerase add to telomeres

GGGTTA repeats
-different versions of the same gene
Point Mutation
-one base change
-can change AA, or may remain same
Insertion or deletion, can result in frame shift
If adenine get's depurinated ....
then you can get thiamine dimers
End-joining Mutations (2)
non-homologous: resulting from helix breakage and repair

homologous: resulting from sister chromatids acting as template for repair
Triplet Expansion: what is it, and what disease?
-CpG repeats cause incorrect adherence during meiosis

Non-disjunction: what is it, and what disease?
-separation in Anaphase 1 leads to uneven separation of chromosomes

Silent Mutation
-single base change, no AA change
Missense Mutation
-single base change, different AA creates
Non-sense Mutation
-single base change, stop codon (UGA, UAG, UAA) created
Frameshift Mutations
-result from in/dels
Transposon Mutations
randomly insert selves into DNA
Exicision Nuclease (and associated disease)
Repairs DNA double strand breaks
-Xeroderma Pigmentosa
One person can have different genes in different cells;
-during development, certain gene mutations can be acquired at different times ... resulting in different cellular karyotypes in same person
DNA Replication (leading and lagging strand)
1) DNA Helicase opens DNA
*topoisomerase 1 prevents supercoiling
2) Primase adds primer
3) DNApol-alpha starts adding dNTPs
4) DNApol-delta takes over

*leading strand; in the direction of opening; continuous addition
*lagging strand; in opposite direction of opening; discontinous addition; RNAase needed to remove primer; DNApol-delta needed to fill in gaps; ligase needed to ligate fragments
DNA pol delta: special ability?
3' to 5' exonuclease ability
adds to lagging strand on 3' end of DNA
(has RNA component that is uses as guide)
Cytarabine: drug mechanism
Good cancer treatment
-inserts arabinose instead of ribose into cytosine nucleotides
-prevents DNA replication
Cyclophosphamide: drug mechanism
-converted to phosphamide in liver
-ALKYLATES DNA, blocking replication
Doxorubicin: drug mechanism
Cancer treatment
-inhibits topoisomerase 2, so no religation of broken strands occurs
Imelestat: drug mechanism
binds RNA template on telomerase
Lynch Syndrome
-mismatch repair system messed up
-leads to HNPCC
Bloom Syndrome
DNA helicase defect
-Okizaki fragment joining impaired
RNA polymerases (differences from DNA pol)
-no exonuclease activity
-no primer needed
-more errors
Which RNA pols make tRNA, mRNA, and rRNA
3: tRNA

2: mRNA

1: rRNA
Transcription: initiation
1) TF2D binds DNA (helicase unwinds)
2) RNA pol 2 binds, get's p-lated, and rapid xC occurs
Alpha-amanitin poisoning
-inhibits RNApol2
pre-mRNA Processing ... (3 things)
1) 5' methyl-guanosine cap
2) splicing by splicosome
3) 3' poly-A tial
RNA splicing
1) Adenine at branch point cuts 5' splice site
2) Free OH at 5' splice site attacks 3' splice site intron
3) lariat shaped intron made!
-presence of cryptic splice site ... leads to incorrect splicing
Chromatin Remodeling Complexes (HATs vs HDACs)
HAT's; histone acyl transferase; transfer acyl group to lysine, making it less positive; less attraction between DNA and histone ... leads to

HDAC's: histine deacetylases; remove acyl groups from histones
What is the role of DNA binding proteins?
-to recruit HAT's or HDAC's
LDL receptor gene regulation
1) SP-1/CRSP always bound to gene
2) SREBP binds SRE, increases HAT's

*this occurs during low cellular cholesterol conditions
-this is partially how statin drugs work
Hydrophobic hormone binding mechanism
1) diffuse through membrane
2) bind to receptor in cytosol
3) taken to nucleus to bind HRE (hormone receptive elements)
Thyroid Hormone Binding Mechanism
1) T3 binds internal receptor
2) taken to nucleus, T3 binds to THR-RXR; causes removal of HDACs, and addition of HAT's
PKA mediated xC control
1) PKA p-lates CREB
2) CREB-P binds CRE
3) Complex binds CBP in nucleus, and HAT's recruited
Rubenstein-Taybi Syndrome
"The Creb-B jew!"

-Creb-BP mutation
-inhibits estrogen receptor

Ribosome Sizes: EuK, ProK, Mito
EuK: 80S (40 + 60)
Mito: 60S
ProK: 70S (50+30)
Ribosome: Small subunit vs large subunit function
Large Subunit: enzymatic activity

Small Subunit: binds the RNA's
xL: initiation
1)eIF-2A binds GTP; complex then binds tRNA-met (ternary complex)
2) Ternary complex then binds small subunit
3) mRNA binds complex
4) Large subunit binds complex
5) eIF2A hydrolyzed
xL: elongation
1) tRNA-met in P site
2) new tRNA comes in with EF-1-GTP
3) complex moves with help of EF-2-GTP
xL: termination
1) Stop codons binds A-site
2) eRF-GTP binds stop codon, gets hydrolyzed
3) complex falls apart
binds small subunit of ribosome
-mimics tRNA by binding A site
prevents peptidyl-bond formation
removes adenine bases
Diptheria Toxin
Inactivates EF-2
N-linked glycoslyation

1) Dolichol-P adds NAGA x 2
2) Mannoses added
3) Compound flipped into ER
4) Dolichol-P adds glycoslate to protein on Asn residues
N-linked glycoslyation; 2 subtypes
1) High mannose
2) Complex

*these modifications occur in the golgi
O-linked glycosylation
-occurs on H-antigen on RBC

-occurs on proteoglycans of ECM
Lysosomal Protein Localization
-must have mannose-P added
-P-lated protein goes to lysosome
Mitochondrial Protein Localization
Needs TOM/TIM complex to guide it
I-cell disease
Lysosomal proteins appear in cytoplasm because mannose-P mechanism is not working
3 Main checkpoints of cell cycle
1) S-phase checkpoint
2) G2/M checkpoint
3) Metaphase/Anaphase checkpoint
G1-CDK and G1/S-CDK target what protein?
S-CDK targets what gene?
1) GF binds
2) Ras activated
4) Increase in MYC
G1 to S-phase transition: mechanism
1) Mitogen binds
2) Cyclin D produced
3) Rb-E2F get's P-lated: Rb-P falls off, E2F acts as TF
4) Cyclin E and A made; CHECKPOINT PASSED
p53 Mechanism
1) UV damage to DNA
2) P53 p-lated
3) Increases p21 (which inhibts G1/S and S cdk's) and p27 (which inhibits CDK's too)
Role of APCs (anaphase promoting complex)
polyubiquinate s and m cdk's

dephos Rb
targets p53 for proteosome degradation
Apoptosis mechanism
1) BAX promotes apoptosis; creates channel in mito
2) Cytochrome C's escape
3) Apoptosome created
4) Caspaces trigger cell death
Oncogene, Proto-oncogene, TSG's: please define
Oncogene: a gene that has been mutated to a state of increased or constant expression ... helps cell cycle move forward

Proto-oncogene: a gene that can become mutated to become an oncogene

TSG: tumor suppressor gene; a gene that stops the cell cycle in order to prevent inappropriate division
Top 3 Causes of Death
1) Heart Disease

2) Malignant Neoplasms

3) Cerebrovascular Disease
What are two mechanisms by which genes get changed into cancer-promoting agents?
1) DNA sequence gets changed

2) Epigenetic changes, including methylation
6 Abilities Cancer Cells need to acquire ...
1) Ability to grow without mitogens
2) Loss of adherance abilities
3) Apoptosis evading mechanisms
4) Insensitivity to contact inhibition
5) Limitless replication (telomerase activity)
6) Sustained angiogenesis
Example of 2 most famous TSG's

Example of 3 Most famous oncogenes
Myc (TF)

Ras (membrane signalling)

Erb-B2 (her2/neu)
Erb-B2 Mutation
aka her2/neu

-constitutively active tyrosine kinase
-doesn't need EGF to grow
Ras signalling
1) Mitogen (GF) binds cell receptor
2) Ras drops GDP, binds GTP
3) Ras-GTP activates RAF
*shut down occurs when GAP hydrolyses GTP
Myc function (and associated cancer)
TF that promotes xC of CDK's

p53 function (and associated cancer)
1) DNA damage occurs
2) p53 gets p-lated, MDM2 falls off
3) p53 act as TF for p21 and p27

*found in Li-Fraumeni
What does HPV target?
Produced E6 and E7

E6: inhibits p53

E7: inhibits p53, p21, and Rb
FAP cancers (and progression)
-colorectal cancer
-characterized by the presence of thousands of polyps

-Defective APC

1) defective APC inherited
2) second allele lost
3) Ras actvated
4) Loss of DCC, then p53
APC function
-TSG; inhibits B-catenins

*B-catenins increase activity of myc and cyclin D
Lynch Syndrome
-don't have as many polyps

-Loss of mismatch repair genes, and microsatelite instability
Malignant (metastatic) cancers have lost ...
adherance capabilities. Loss of E-cadherins (TSG)
Metastasis: invasion
-use serine proteases to break down collagen
Philadelphia Chromosome: defect, which cancer?
-translocation of ABL from chromosome 9 to chromosome 22; comes under regulation of BCR gene
MAB that binds her2/neu receptors; causes internalization
-targets CD20+ in non-hodgkins lymphoma
-targets cells for destruction
Ras Signaling: Normal and Mutant
Normal: Ras bind GTP; Mapkkk sequence, c-myc activity increased

Mutant: loses GTPase activity
Burkitt's Lymphoma cause
-balanced translocation of myc
Li Fraumeni Syndrome
-Loss of p53 function
Treatments for Rb (3)
1) Vincristine -inhibits microtubule
2) Carboplatin - alklylated DNA
3) Etoptoside - inhibits topo iso 2
FAP inheritance and defect
-on APC gene (TSG);
-autosomal dominant
APC normal function
-inhibits B-catenins
-B-catenins upregulate myc and D-cyclins
FAP progression
1) Inherited FAP
2) 2nd FAP mutation
CML cause
-The philadelphia chromosome
-ABL (oncogene) moves to BCR promoters area
Imantinib Mesylate (Gleevec)
TKI inhibitor: CML
Breast Cancer treatments (3)
1) Herceptin: binds EGF receptor (her2neu)
2) Leptanib: TKI inhibitor
3) Gefitinib: EGF inhibitor
MAB targeting CD20+ cells;
-treatment for non-hodgkin lymphoma; targets cell for destruction
DNA intercolating agent that decreases the activity of topoisomerase II
Naladixic Acid
inhibit bacterial topoisomerase 2
inhibit bacterial topoisomerase 2
Sickle-Cell Disease
-E to V mutation at AA#6; results in abnormal beta chain; sickle cell formation
Triplet Expansion found in what disease?
Non-disjunction found in what disease?
Down's Syndrome
Xeroderma Pigmentosa
-loss of excision endonuclease
-Cancer treatment
-In cytosines, it inserts an arabinose in place of ribose
-converted to phosphamide in liver
-acts as alkylating agent of DNA
-intercolating agent, blocks topoiso2 action
binds RNA template on telomerase
Lynch syndrome defect in ...
mismatch repair
Bloom syndrome defect in ... find what?
DNA helicase; results in increased okisaki fragments
When RNA pol 2 gets P-lated, what happens?
Other TF's fall off (TF2D) and xC goes faster
Poison of the death cap mushroom;
-inhibits RNA pol 2
Inhibits RNA pol 2 in gram + bacteria
-can induce cP450's
What does LDL receptor bind,
-Binds Apo B, E on LDL and VLDL

1) CRSP-SP1 always bound on LDL gene
2) SREBP-SRE bind CRSP-SP1; increase HATs!
Cortisol mechanism
1) Binds receptor inside cell
2) Dimerizes
3) Binds to nucleus, acts as TF
PKA cell signaling
1) CREB gets P
2) Creb-P binds CRE
3) Creb-P-CRE bind to CBP
4) Increased HATs!
Creb Binding Protein defect
-estrogen receptor inhibitor
-still binds nucleus but no XC
binds small subunit of 16s ribosome in bacteria
AB: mistranslation of codons
blocks A-site (mimics tRNA)
prevents peptidyl bond formation
inactivates ribosome; hyrolyzed adenine on rRNA
Diptheria Toxin
Inactivates EF2 by adding ribose to dipthamide
Cystic Fibrosis Defect
-CFTR doesn't get properly glycated
I-cell disease defect
Mannose-P not present, so you don't get localization into lysosome
Ubiquinone amide bond
Creates amide bond between glycine on UBQ and lysine on protein
In N-linked and O-linked glycoslyation, where do sugars add to on proteins?
N-linked: AsN

O-linked; serine/threonine
Top 3 Causes of Death
1) Heart Disease
2) Malignant Neoplasms
3) Cerebrovascular disease
Cancer History
First record of cancer: 1600 BC

Hippocrates: used "carcinomas" for crab, due to the angiogenesis

Galen: used 'oncos' for swelling
Cancer definition
Genetic disease of the somatic cells characterized by uncontrolled cell division.

-Activation of an oncogene, or deactivation of a tumor suppressor
Tumor Formation
Changes in DNA sequence, and/or changes in methylation
-mutated form of genes (proto-oncogenes) that stimulate cell division
Tumor Suppressor Genes
Genes that control cell division, DNA repair genes, or apoptosis genes
6 Abilities Cancer cells need to require to become metastatic
1) Self-sufficiency in growth signals
2) Insensitivity to anti-growth signals (no contact inhibition)
3) Evading apoptosis
4) Limitless replicative potential
5) Sustained angiogenesis
6) Tissue invasion and metastasis
Genetic Instability
Look at images
Proto-oncogenes and cell cycle
Act as accelerator; drive cell cycle faster: promote growth
Tumor suppressor and cell cycle
Act as brakes: elments that stop, regulate, or control the cell cycle
Oncogenes and cell cycle
Mutated version or normal gene; WORKS TOO WELL; doesn't respond to normal cell signals to stop
List of Oncogenes
Erb B2 (her2/neu): growth factor receptor

Ras: signal transduction gene

Myc: nuclear protein
Deletion/Point Mutation
results in hyperactive protein
Regulatory Mutation
normal protein greatly overproduced
Gene Amplification
Normal protein overproduced due to multiple copies of gene
Chromosome rearrangement
Gene is now under control of the wrong promoter;
-over-production of normal protein
Erb B2 Mutation Process

-encodes truncated epidermal growth factor receptor (tyrosine kinase). CONTITUTIVELY ACTIVE without EGF around

-found in certain breast cancers
Ras: normal function
1) Growth factors bind receptor, activate GRB2-SOS
2) GTP binds Ras, GDP falls off; RAS ACTIVE
3) Ras-GTP activates RAF
4) GAP activates (GTPase activating protein)
5) GTP to GDP, Ras inactive
Ras: oncogenic function
Point mutation: Ras loses GTP-ase activity; remains ACTIVE!!!
Genes with loss of xC control
myc, fos, jun

-DNA physical change or methylation of promoters
TF ... leucine zipper domain
-dimerizes with MAX

Upregulates xC for activation of CDKs
Burkitt's Lymphoma
Balanced translocation of myc:

-myc starts to be under control of wrong promoters
-gets translocated to immunoglobulin gene;
-found in B-CELLS!!!
-act as brakes

-cell division controlling genes
-DNA repair genes
-Apoptosis genes
p53: normal
normal: acts as TF;
1) DNA damage
2) p53 gets phosphorylated, MDM2 falls off
3) Active p53 acts as TF for DNA repair and apoptosis
p53: it's effects
1) upregulated p21
2) too much DNA, BAX activated
p53: In cancers
Found in over 50% of colorectal tumors
Li-Fraumeni Syndrome
-autosomal dominant: p53 loss of function

-by age 30, 50% of carriers will develop one malignancies
-15% develop a second cancer
-cancer occurs usually before 45
-cells that replicate most are most susceptible;
Cervical Cancer
HPV targets p53
HPV and cervical cancer
Produces two early genes

E6 - inhibits p53
E7 - inhibits p53, p21, and Rb
"Two-hit model"
Hit 1: inheritance of a mutation of Rb1 gene
Hit 2: somatic mutation that mutates the other function RB1 gene
Loss of function of RB ...
... means E2F remains free.
-upregulates cell cycle
Retinoblastoma Cancer
Rare (1/20,000)
Two forms: Genetic (bilateral, both eyes) or Sporadic (new mutation)- unilateral
Why does Rb occur in retina?
Retina is MOST metabolically active tissue in the body;
-retina in aggressive are (UV, ROI's)
Treatments for RB
laser treatment to stop angiogenesis

-chemical inhibitors
Rb treatment
microtubule inhibitor
alkylating agent

Rb treatment
inhibits topoisomerase 2

Rb treatment
Colorectal Cancers (2 common)
FAP (familiar adenomatous polyposis)

HNPCC, Lynch Syndrome
FAP: overall
-autosomal dominant
-effects adenomatous polyposis coli gene (nonsense and frame shifts)

APC is tumor suppressor

-Develop thousands of polyps
APC: normal
Increase Beta-catenin

B-catenin acts as TF for myc and cyclin D

Normal state: APC binds, UBQnd B-ccatenin for degradation
FAP tumor progression
1) Patients have hereditary APC allele
2) Loss of second allele leads to lots of polyps (adenoma)
3) Ras activated (more adenoma)
4) Loss of DCC (lots of adenomas
5) Loss of p53 (carcinoma)
Lynch Syndrome
-80% chance of developing colorectal cancer
-5% of all colorectal carcinomas

Cause: defect in mismatch repair genes

Loss of heterozygosity
Telomere Length and Cell-life Span
Telomerase needed for DNA pol to replicate ends to telomere

-increased presence of telomerase in cancer cells
1) Breaks down the extracellular matrix
2) Enter lymphatic or blood vessels
3) Exit and grow in suitable distant site
Difference between benign and tumor cells
Malignant have less adhesiveness
-down-regulate E-cadherins
How do cancer cells invade ECM?
Make enzymes to degrade collagens, glycoproteins, proteoglycans
-serine proteases
-matrix metalloproteinases
Intravasation: enter circulation

Extravasation: move towards new tissue
Epigenetic Changes
-DNA sequence unchanged, but the gene is highly methylated

-ex TSGs: methylated, xC shutdown
Hypomethylation in which cancers?
colonic neoplasms, Wilms tumor, breast/ovarian cancer
Hypermethylation in which cancers?
small cell lung cancers, RB, Histone modifcation
CML (chronic myeloid leukemia)
Philadelphia Chromosome: BCR and ABL(tyrosine kinase) genes;
-creates new protein!!! enhanced kinase activity, constitutively expressed!!!

-under control of BCR promoter
3 Phases of CML
Chronic Phase: 5 yrs, mild symptoms
80% survival with treatment

Accelerated Phase: .5 to 1 yr; fever, bone pain, spleen and liver growth
50% survival with treatment

Blast Crisis: Poor diagnosis
-3-6 months
20% survival with treatment
Imantinib mesylate (GLEEVEC)
-tyrosine kinase inhibitor (TKI)
NSCLC: overall
non-small cell lung cancer

-smoking increases with amount/duration
-asbestos also increases

Treatment: TKI's of EGF receptors
Breast Cancer - Targeted Treatment
-25% have overexpression of Her2 - tyrosine kinase growth factor receptor
-monoclonal antibody; binds receptor, causes internalization; reduces amount of receptor available
TKI for triple positive cancers; prevents P-lation of receptors
treat CD20+ non-Hodgkin's lymphoma
-MAB that targets the CD20 surface marker; targets it for destruction
How to MAB's work?
1) Can block receptor
2) Can signal apoptosis
3) Can recruit NK cells
4) Can be conjugated to toxins (doxorubicin)
Gefitinib Action
EGFR inhibitor
-used for breast cancer
Which cancer has mismatch repair and microsatelite instablility?
Lynch: HNPCC:

-microsatelites are short repeats in genes that are prone to error and get fixed by DNA mismatch repair
BCL-2 Mutation
Anti-apoptosis gene: gain of function in cancers
Fos, Myc, Jun
Proto-oncogenes that act as TFs
Four stages of cell cycle? Which are interphase?
G1, S, G2 =interphase

M phase=mitosis
What are the 3 big checkpoints?
1) Start Checkpoint: environment favorable?
2) G2/M checkpoint: all DNA replicated? Environment favorable?
3) Metaphase/Anaphase checkpoint: all chromosomes attached to spindles?
What are the roles of cyclins/CDK?
-cylins activate CDK's; later are destroyed by UBQ
-CDKs are serine-threonine protein kinases; P-late genes
Cyclin D: parter, and complexed

Cyclin E

Cylcin A
CDK-2 (1)

Cyclin B

Which cyclins present during cell cycle?
G1: G1-CDK and G1/S-CDK

S and G2: S-CDK

DNA damage inhbits which complexes?
G1/S-CDK and S-CDK and M-CDK
Unreplicated DNA stops what complex?
G1-CDK and G1/S-CDK target what protein?
S-CDK targets what gene?
What inhibits RB and E2F
p21, 27
How do growth factors work?
Ex. EGF PDGF; increase cell proliferation
1) bind receptor 2) dimerization and cross-p-lation via TYROSINE KINASES
Please describe the Ras-cascade;
1) GTP binds Ras ... Ras is Active
2) Active Ras increases Map-KKK
3) ATP used: MAP-KK activated
4) ATP used: Map-K activated
5) MAP-K P-lates regulatory proteins; INCREASES MYC!
Mechanism: G1 to S
1) Mitogen binds
2) Cyclin D increased
3) Rb-E2F gets P-lated: P-lated Rb is inactive
4) Free E2F allows for xC of S-phase genes
5) Cyclin E and A created
P53/P21 Mechanism
1) UV Damage to DNA
2) P53 P-lated
3) P-53 increases P21(inhibits G1/S-CDK and S-CDK) and P27(increases CDK-I's)
Mechanism: S to G2
-spindle proteins made here in G2

1) P-lation of ORC (original replication complexes) leads to replication
Mechanism: G2 to M
1) M-CDK P-lates nuclear laminas ... spindle app forms
2) chromosomes condense, envelope breaks down
3) CDC20 increases APC's ... sister chromatids seperate
Role of APCs
Poly-ubiquinate S and M CDKs

Dephosphorylates Rb
What is MDM2?
Targets P53 for proteosome-mediated degradation
What can cause apoptosis?
p53, TNF-R, Fas
Apoptotic Mechanism
1) BH3 only protein triggered ... activates BAX
2) BAX creates channel into mitochondria
3) cytochrome C's excape
4) Create apoptosome
5) Increase caspase 9; this increases executioner caspaces (3,6,7)
Compare Total Size: Euk cytoplasm ribosome, mitochondrial ribosome, and bacteria
Cytoplasm: 80S
Mito: 60S

Bacteria: 70S
Small subunit size differences
Cytoplasm: 40S (18S RNA)
mito: 35S (19S RNA)

Bacteria: 30S (16S RNA)
Large subunit size differences
Cytoplasm: 60 S (25S RNA)
Mito: 45S

Bacteria: 50S
Main function of small/large subunit
Small: binds RNAs (tRNA, mRNA)

Large: catalytic activity (peptide bond former)
Euk/ProK mRNA comparison
Euk mRNA is monocistronic (just one protein made from the strand)

ProK is polycistronic (multiple ORFs, multiple proteins from same strand)
How do tRNA's work?
-have 3 base anticodon at end
-have amino acid linked at acceptor terminus

-has inosine; can pair with A, C, or U!!!
xL: Initiation
1) EIF2A (eukaryotic initiation factor) activated by binding GTP
2) EIF2A-GTP binds initiator tRNA-met to form TERNARY COMPLEX
3) Ternary complex binds small ribosomal subunit
4) mRNA binds to complex
5) Large subunit binds, EIF2A hydrolyzed to GDP; released; PREINITIATION COMPLEX COMPLETE
xL: Elongation
1) tRNA met bound to P-site of ribisome
2) 2nd AA-tRNA placed into A site; EF-1-GTP binds; gets hydrolyzed
3) Peptidyl bond formed
4) Ribosome moves one codon down
5) EF-2-GTP binds, gets hydrolized; A-site is empty
6) next AA comes in
xL: Termination
1) One of the stop codons binds the A-site
2) eRF-GTP (release factor) pairs with stop codon
3) eRF-GTP gets hydrolized, peptide is released from P-site
4) Ribosome separates
binds small subunit; prevents initiation; wrong protein made
mistranslation of codons
blocks A-site (mimics tRNA)
prevents peptidyl-bond formation
RIP: ricin inactivating protein; removes adenine bases (acts as enzyme)
Diptheria Toxin
adds ribose from NAD to EF-2; inactivates EF-2

xL Regulation
1) proteins can bind 5'UTR, masking start codon

2) P-lation of eIF-2A can inactivate it

3) Use of IRES (internal ribosomal entry sites)
-xL can start on uncapped mRNA
Chaperones: mechanism
-bind ATP; take misfolded protein and loosen it up

-allow protein to refold itself correctly
Exported Proteins; Mechanism
1) emerging peptide on ribosome has signal sequence; xL stops
2) SRP (signal recognition particle) binds signal sequence
3) Complex binds a translocon via docking protein
4) Peptide continues xL into ER lumen
5) Signal peptidase (on lumen wall) cleaves SRP off of peptide
Unfolded Protein Response
1) Accumilation of unfolded proteins ... UPR
2) xL stopped; HSP's (chaperones) made
3) If inadequate, then APOPTOSIS
Glycosylation: specificity
-specific to donor, acceptor, and linkage (O or N)
N-linked Glycosylation Mechanism
ADDED TO PROTEIN IN ER: b-1,4 linkages

1) Dolichol-P adds N-acetyl-glucosamine via UDP-transferase to ITSELF
2) 2nd NAGA added
3) Mannoses added
4) Compound flipped into ER
5) Dolichol-P donates glycoslation molecule to ASN on peptide
Two types of N-linked glycosylation
More modification occurs later in the golgi

1) High Mannose
2) complex (galactose and sialic acid added)
O-linked Glycoslation Mechanism

1) Glycosyltransferases add to fully folded proteins at serine or threonine residues in GOLGI
What are two types of O-linked peptides?
Proteoglycans of ECM and H-antigen on RBC
Congenital Defects in Glycoslation: 2 types
1) CDG-1; defective synthesis of lipid-linked oligosacharide precursor

2) CDG-2: defective trimming of disacharide chains
Of proline
gets hydroxylated
Of lysine
gets acetylated
Of cysteine
can create formyl-glycine
N-terminal trimming
removing methionine
Hydrophibic entity additions
1) Myristic acid to N term
2) Palmitic Acid to cysteine
3) Prenylation of cysteine at C term
GPI anchor (hydrophobic)
inositol anchor added to C term
How does protein get localized to lysosome?
1) UDP-mannose x2 added to protein
2) Mannose falls off
3) P-lated protein goes to ribosome
How is protein moved to the mitochondria?
1) Presequence on peptide allocates peptide for mitochondria
2) HSP70 orients protein properly
3) Moved into mito through TOM and TIM
4) HSP-60 guides folding in matrix
Cystic Fibrosis
CFTR-1 not glycated properly; gets degraded
I-cell disease
surface mannose residues not P-lated; lysosomal proteins appear in cytoplasm and serum

-lysosomes appear dense
Protein Degradation: Lysosomal Mechanism
1) Autophagy: cytosol protein enters lysosome, taking some cytoplasm with it
2) Endocytic: clathrin pit create, clathrin vessicle fuses with lysosome; peptide degraded
Protein Degradation: Proteosome
1) Protein gets highly ubqn'ed
2) degraded by proteosome
Ubiquinin reaction
Mechanism: C-term glycine on UBQ creates amide bond with lysine on peptide
Types of Ubiquination
1) Mono: regulate a protein (like, histones)

2) Poly: degradation
What determines how long a protein will survive in cells?
1) Misfolding and PEST shorten lifespan
2) Arg/Lys rich proteins; shorter lifespan
3) Ser/Met rich proteins; long lifespan
Differences in RNA Pol (from DNA Pol)
RNA pol has no exonuclease activity (no proofreading)
RNA pol does not need primer
More errors
Which RNA pols make mRNA, tRNA, and rRNA
mRNA: pol 2

rRNA: pol 1 (3)

tRNA: pol 3
What is concensus sequence?
set of promoters, enhancers upstream of start site;
-most common nucleotides found at this area
-includes TATA box
Transcription: INITIATION
1) TF2D binds DNA
2) TF2H (helicase) attaches
3) DNA opened at A-T rich regions
4) RNA pol 2 binds (slow TC)
5) C-terminus of RNA-pol get's p-lated, other TF's fall off,
6) Elongation complex now rapidly does xC

*chromatin remodeling complex part of RNA pol complex
Typical Layout of Gene
_____enhancer____gene specific elem____CAAT or GC-rich _____TATABOX
-found in death-cap mushroom

Mechanism: 1) eat DC mushroom 2) increase in alpha-amanitin levels

-alpha-amanitin inhibits RNA pol II
inhibits RNA Pol 2 in Gram (+) bacteria
-can induce cP450's in humans
pre-RNA processing (3 things)

1) Capping
2) Splicing
3) Poly-adenylation
RNA capping
-methyl-guanosine added to 5' end
-distinguishes mRNA; protects against degradation
RNA Splicing (3 steps)
Done by splicosomes (protein +snRNA)

1) Adenine at BRANCH POINTS cuts 5'splice site intron end
2) Free OH on exon attacks 3'splice site intron end
3) lariat intron made + mRNA
1) small poly-A tail (or GU/U rich tail) is on mRNA 3'
2) That part is cleaved, and replaced with 200 A's
RNA Nuclear Export
mRNA + exportin + CBC (cap binding complex): mRNA transported into the cytosol
Alternative Splicing
Different introns can be removed; different proteins can be made from the same gene
-mutation of HbB gene

B-chain of Hb deformed

B(0) mutant: normal acceptor at intron 2 destroyed ... cryptic splice site : no b-chain made

B(+) mutant: new acceptor siteon intron 1 ... leads to no B-chain
What are 2 types of chromatin remodeling complexes?
HATs (upregulate xC) and HDACs (downregulate xC)
histone acyl-transferases

-acetylate lysine on histones; less (+) charge
-less attraction between histone and DNA

histone deacytlases

-remove acetyl group from histones: more (+) charge: more attraction between histones and DNA

What can happen at N terminus of histone?
-acetylation, methylation, P-lation
What happens if histone is methylated?
-more HDAC's bind
3 classes of DNA binding proteins: types, actions?
Bind DNA, recruit HATs or HDACs

1) Helix-turn-helix

2) Zinc finger proteins

3) Leucine Zipper Motif
2 alpha helices connected by short AA

-side chain of AA recognizes DNA
Zinc-finger proteins
alpha helix + zinc

-alpha helix binds DNA
Leucine Zipper Motifs
two alpha-helices form dimer

every 7th AA is leucine
LDL receptor gene
-codes for an integral membrane protein
-Recognizes ApoB, E on LDL and VLDL
How is LDL receptor gene regulated?
In low cellular cholesterol conditions ...

1) Basal factors are always bound to gene: SP-1, CRSP
2) SREBP-1A binds to SRE ... increases HAT
3) This increases xC speed!

In normal cellular cholesterol conditions ...

1) SP-1and CRSP bound to G-C rich regions; slow xC
Overall: how do hydrophobic hormones work?
Ex. Thyroid hormone, cortisol

1) Bind receptor inside cell
2) Transported to nucleus
3) Hormone receptor complex bind HRE's (hormone responsive elements)
Glucocorticoid Responsive Element
Estrogen Responsive Element
Cortisol Binding Mechanism
1) Cortisol binds internal cell receptor
2) receptor changes conformation, releases inhibitory subunits
3) Receptor-hormone complex dimerize
4) Dimer imported into cell, bind GRE
5) Increase genes (PEPCK, ex)
Throid homrone mechanism
In absence of thyroid hormone, you have basal TF bound to HRE: THR-RXR (retinoid x receptor) bound to HRE; HDAC's bound

1) Thyroid hormone comes in, binds THR/RXR;
2) HDAC falls off, HAT binds
3) xC!!!
PKA mediated xC control Mechanism
1) Hormone binds G-protein receptor
2) Increase in PKA
3) CREB gets P-lated
4) CREB-P binds CRE
5) CREB-P-CRE binds CBP; allows EP300 cofactor bind
6) Complex has HAT activity
Rubinstein-Taybi Syndrome
-causes hypoplastic maxilla, retardation
-Autosomal dominant

Cause: Mutation in Creb-BP (CBP)
Estrogen hormone Mechanism
1) enters cytoplasm, binds receptor; inhibitory proteins fall off
2) Estrogen+R go to nucleus, bind ERE
3) Complex attracts cofactors, increase xC
*alpha and beta estrogen receptors exist
How does tamoxifen work?
Competitive inhibitor of estrogen receptor; receptor+tamoxifen still bind nucleus, but NO xC occurs
cMYC: type of protein domain
Helix-turn-helix; proto-oncogene
A different type of the same gene: eye color allele; etc etc

-variants of a gene with a slightly different sequence
Genetic Polymorphism
many different alleles for the same gene
Missegragation of chromosomes; how common?
occurs 1/100 cell divisions
Rearrangement of chromosome (faulty recombination): how common?
1/60,000 cell divisions
Gene Mutations (one nucleotide change)
1/10(10th) cell divisions
Germ Line vs Somatic Mutation
Germ line: can be inherited

Somatic line: not heritable
Point Mutation
-one NA changes
-demethylation/deamination of base
-methylation of cytosine
Insertion/Deletion mutations
-incorrect recombination during meiosis
-strand slippage
-results in frame-shift
What bases does deamination result in?
Hypoxanthine, Xanthine, and Uracil?
What results if cytosine deaminated?
Uracil forms; Uracil DNA glycosylase turns it back into cytosine
What results if adenine depurinated?
results in apurinated thymine; if UV hits, it can cause dimers
End-Joining mutations (2 categories)
non-homologous: 1) helix breaks 2) nucleotides removed 3) strands joined
-DNA deleted!

homologous: during division; sister chromatid can guide reassembly of nucleotides
Triplet Expansion Mutations: what disease?
-due to constant repeats, leading to strand slippage
-can result in loops structure (meiosis)

If loops kept, DNA gained
If loops lost, DNA lost

Found in people with Huntington's
Non-disjunction Mutation
Occurs in Anaphase 1:
uneven separation of chromosomes

-found in Down's Syndrome
Silent Mutation
-Single base change
-No AA change
Missense Mutation
-Single base change
-Different AA created
Non-sense Mutation
-single base change
-early stop codon created (UAA, UGA, UAG)
Frameshift Mutation
Deletions or Additions
-can result in different AA's, stop codons, etc
Transposon Mutation
Genes moved around, inserted randomly (usually into introns)
How do DNA double strand brakes arise? How do you fix?
Through ionizing radiation (X-ray)

1) Dimer recognized; helicase binds DNA
2) Excision Nuclease cleaves DNA (30bp)
3) DNA Pol and Ligase fill in the gap

-usually leads to loss of DNA
Xeroderma Pigmentosa
Extreme sensitivity to sunlight, pigmentation changes, skin cancers

-due to lack of excision nuclease
Mosaicism (and example)
One person, but they have different genes in different parts of their body;

Trisomy 21: during embyology, one cell gives rise to daughter cell with no copies of 21, and itself has 3; this is one way of getting trisomy; other ways exist
Origins of Replication: what occurs?
1) DNA helicase unwinds DNA
*topoisomerase I cuts one strand, P-lates base to prevent supercoiling
2) Primase creates primer
3) DNA pol-alpha beings adding NTP
4) Sliding clamp added, DNA pol-delta comes in and continues adding NTPs
Leading Strand
Moves in the direction of opening replication fork

Lagging Strand
Okisaki fragments made; RNAase removes primers
-DnA pol delta fills in gaps
-DNA ligase ligates DNA gaps
DNA Pol Delta: what's special?
has 3' to 5' exonuclease activity (proofreading)
Strand-directed mismatch repair
New DNA might have nicks and gaps due to errors;
-repair enzymes can fix this using the original strand as template
-linear molecules, gets shorter every time you divide
-GGGTTA repeats
-replicated by TELOMERASE
Telomerase mechanism
1) Sees incomplete lagging strand in telomere region
2) uses RNA template to complete lagging strand
blocks DNA replication
-inserts arabinose instead of ribose into cystidine
converted to phosphamide in liver
-alkylates DNA, resulting in crosslinking
-blocks replication
-cancer treatment
-prevents religation of broken strands by blocking Topoisomerase 2
bind RNA template on telomerase
reactivate telomerase in CD4+ cells
Lynch Syndrome
-Hereditary non-polyposis colorectal carcinoma
-patients have 80% chance of getting colorectal cancer

Bloom Syndrome
-dwarfism, photosensitivity, big nose

DNA helicase defect
What happens if methylated cytosine gets converted to thymine?
-Then DNA repair enzyme will have hard time distinguishing which is correct base: 50% chance
If thymine dimer occurs ...
then you have p53 activation, increase POMC, and increase melanin
Balanced/Unbalanced Mutations
Balanced: no genes lost

Unbalanced: genes and DNA lost
single nucleotide polymorphism: 1 base change in DNA; account for most of the differences in people
Main cause of aneuploidy?
Maternal Age: Sperm eggs remain in arrest for 45 years ... older the mother, the higher the chances of chromsomes separating badly
Which carbon atoms of the ribose are linked to phosphates?
3' O-P link and 5' C-O-P link
Where are bases attached to ribose?
What's the diff between ribose and deoxyribose?
Ribose: OH at 2' position

Deoxyribose: H at 2' position
How do you calculate percentage of AA's?

What are the B, A, and Z forms of DNA?
B-form: right handed helix, 10.4 base pairs per turn (most DNA)

A-form: right handed, 11 bp per turn; found in DNA-RNA hybrid or RNA ds

Z-form: left-handed helix stabilized by methylated cytidine bases
3 DNA stabilizing forces
1) H-bonding between bases
2) Hydrophobic/stacking interactions of base-pairs
3) Interaction of polyanionic backbone with cations
What is supercoiling?
Condition DNA is in when it is not getting xC
Negative/Positive supercoiling
DNA opens up helix, unwinds (negative supercoiling) causes positive supercoiling upstream and downstream
DNA Topoisomerase I
cuts one strand of DNA double helix, allows other to rotate, and then gets religated
DNA Topoisomerase II
Manipulates two in-tact double helices; allows other helix to pass through; later, religates
How many genes, nucleotides, chromosomes in human genome?
Genes: 25,000

Chromosomes: 46

Nucleotides: 3.2 x 10(9th)
Categories of non-coding DNA
tRNA, siRNA, miRNA, rRNA:

enhancers, promoters, etc etc
Single Copy DNA vs Multiple Copy DNA
Single Copy: make proteins, make RNA, spacers (introns)

Multiple Copy: Satelite DNA, and Dispersed Repetitive DNA (LINES and SINES)
What's special about dispersed repetitive DNA?
-LINES and SINES can cut and paste themselves in random parts of the genome
Gene definition
DNA sequence that is transcribed to make to eventually lead to a protein or an RNA molecule
Exons: expressed, can create protein

Introns: cut out by splicosome; can serve regulatory purpose
What has most introns? Least?
Dystrophin gene: 99% Introns

Histone H4: no introns
What are gene families? How do they arise?
Genes that have similar AA sequences, but yield different protein;
Arise from unequal cross-over events in meiosis
A gene that once coded for a protein, but is now silenced by a better version of the gene;
-a duplicate gene
Chromatin composition
1/3 DNA, 2/3 proteins (histones)

a) DNA + protein= histone
b) histones wrap around each other = solenoids
c) solenoid gets looped = 300 nm looped domains
What are the structures of nucleosome? Charge of DNA and histones?
DNA + H1 and 2(H2A, H2B, H3, H4)
-DNA: - charged
-histones: + charged AA's
Function of centromere
Where mitotic spindle attaches ... during cell division
Mitochondrial Genome Differences (3)
-circular molecules
-multiple different copies
-encode 13 mitochondrial proteins, 24 RNA's
Benefit of having nucleus
1) allows for separate areas for xC and xL
2) controls access of regulatory proteins, viruses, enzymes etc
Light stain in micrograph
-loose DNA; allows xC
Dark stain in micrograph
-tight DNA: no xC
Big, large dark stain in micrograph
-xC of rRNA
Active transport mechanism into nucleus (TFs)
1) Importin binds protein outside in cytosol: shuttled into Nucleus
2) Ran-GTP binds importin; protein released into nucleus;
3) Ran-GTP-Importin transported to cytosol
4) RanGTP (Ran-GAP) RanGDP, and importin goes free
Active transport out of nucleus (RNA, proteins)
1) exportin binds RNA or protein in nucleus
2) Ran-GTP binds exportin-RNA complex
3) Exported to cytoplasm
4) Ran-GTP complex (RanGAP) Ran GDP + RNA + exportin
inhibits topisomerase II: cancer treatment
Nalidixic acid
inhibit bacterial topoisomerase II
inhibit bacterial topoisomerase II
What is an intercolating agent?
wedges itself into DNa; opens space;
-EthBromide is one of these
can cause alternate splicing in same gene
Sickle-cell Disease
B-globulin gene: E to V change at AA#6: HbS creates polymers

Treatment: hydroxy-urea (reactivates HbF)
A chromosome map: shows all chromosomes
Fluorescent in-situ hybridization:
-allows individual chromosomes to be stained
3-strand DNA structure: prevent digestion by enzymes; on ends of DNA