Biochem 1 Flashcards
What makes up a nucleosome?
Negatively charged DNA loops twice around positively charged histone octamer.
Histones have a lot of what?
Rich in lysine and arginine
Purpose of H1
Binds to nucleosome and to linker DNA to stabilize the chromatin fiber: it is the only one NOT in the nucleosome core.
DNA and histone synthesis during what phase of mitosis
S phase
Nucleosome core histones
H2A, H2B, H3, H4 (each x2)
Heterochromatin
Highly condensed, not active
Euchromatin
Transcriptionally active
DNA methylation in prokaryotes
Template strand Cs and As are methylated to allow mismatch repair enzymes to distinguish parent from daughter strand.
DNA methylation in eukaryotes
CpG islands to repress transcription
What are CpG islands exactly?
Cytosine next to Guanine in a strand of DNA. The cytosine can be methylated, in fact most of the cytosines in CpG islands are.
Histone methylation
Usually reversible, represses transcription, but can occasionally activate it.
Histone acetylation
Relaxes DNA coiling, increasing transcription
What are the pyrmidines
Pyrimidines CUT
Thymine vs Uracil
Thymine has a methyl, uracil is a deaminated cytosine
Amino acids necessary for purine synthesis
GAG-Glycine, Aspartate, Glutamine
What makes up pyrimidines?
Carbamoyl phosphate and aspartate
Nucleoside vs. nucleotide
nucleoside is base + sugar, -tide has 3’-5’ phosphodiester bond linked phosphate(s)
Basic steps of Purine synthesis
- Star with sugar + phosphate (PRPP) 2. Add base
What is PRPP?
Phosphoribosyl pyrophosphate (it has a phosphate instead of a base attached to the ribose)
Basic steps of Pyrimidine synthesis
- Make temporary base (orotic acid) 2. Add sugar + phosphate (PRPP) 3. Modify base
What turns ribonucleotides to deoxyribonucleotides
Ribonucleotide reductase
Carbamoyl phosphate used in what metabolic pathways
De novo pyrimidine synthesis and the urea cycle
Purine base production steps
- Start with Ribose 5-P
- Turn to PRPP by PRPP synthetase
- Produce IMP through some steps
- AMP and GMP produced (GMP by IMP dehydrogenase?)
What does de novo purine synthesis require?
Aspartate, glycine, glutamine, and THF
Pyrimidine base production steps
- Combine Glutamine and CO2 with Carbamoyl phosphate synthetase II to produce carbamoyl phosphate (uses up two ATP)
- Carbamoyl phosphate + Asparate to produce Orotic Acid
- Orotic acid + PRPP to produce UMP
- UMP to UDP
- UDP to CTP or dUDP with ribonucleotide reductase
- dUDP to dUMP
- dUMP to dTMP by Thymidylate synthase
- Tetrahydrofolate in N5N10methyleneTHF is what is used to add the methyl group.
Draw out the pathways!
Writing them out isn’t very useful.
Leflunomide target
Inhibits dihydroorotate dehydrogenase
Mycophenolate and ribavirin target
Inhibit IMP dehydrogenase
Hydroxyurea target
Ribonucleotide reductase
6-mercaptopurine (6-MP) target AND its prodrug
Prodrug is azathioprine. They both inhibit de novo purine synthesis.
5-fluorouracil (5-FU) target
Inhibits thymidylate synthase (lowers deoxythymidine monophosphate (dTMP))
Methyotrexate (MTX), Trimethoprim (TMP), and pyrimethamine target
Inhibits Dihydrofolate reductase (lowers dTMP) in humans, bacteria, and protozoa, respectively. (MTX in humans, TMP in bacteria, Pyrimethamine in protozoa)
Does myophenolate/ribavirin only affect GMP production?
Yes, for de novo GTP production.
Guanine to Cuanylic acid (GMP)
HGPRT + PRPP
Hypoxanthine to Inosinic acid (IMP)
HGPRT + PRPP
Adenine to Adnylic acid (AMP)
APRT + PRPP
Adenosine to Inosine
Adenosine deaminase (ADA)
Hydroxanthine to Xanthine
Xanthine oxidase
Xanthine to uric acid
Xanthine oxidase
Draw out the purine salvage deficiencies
…
Adenosine deaminase deficiency path
Excess ATP and dATP imbalances nucleotide pool via feedback inhibition of ribonucleotide reductase leading to the prevention of DNA synthesis and thus lower lymphocyte count
Adenosine deaminase deficiency and what disease
Autosomal recessive SCID (one of the major causes)
Lesch-Nyhan path
Defective purine salvage from absence of HGPRT. Excess uric acid production and de novo purine synthesis.
Lesch-Nyhan genetics
X-linked recessive
Lesch-Nyhan presentation
Intellectual disability, self-mutilation, aggression, hyperuricemia, gout, dystonia.
Lesch-Nyhan tx
Allopurinol or febuxostate (2nd line)
Lesch-Nyhan Mnemonic
HGPRT: Hyperuricemia, Gout, Pissed off (aggressin,self-mutilation), Retardation, Dystonia
What is HGPRT?
Hypoxantine-Guanine Phosphoribosyltransferase
Degenerate code
Multiple codons for most amino acids
Methionine codon
AUG
Tryptophan codon
UGG
What is commaless, nonoverlapping code?
Commaless means that no codons are used as punctuation, it is read straight through (at least the exons, etc.). Nonoverlapping means one codon in a sequence leads to one amino acid. In viruses, the genes can overlap.
Universal code exception
Mitochondria in humans. The codons can be a little different.
Origins of replication in prok. and euk.
Prok. have 1! (theta-replication)
Euk. have multiple (large chromosomes)
Single-stranded binding proteins
Prevent strands from reannealing
DNA topoisomerases
Create single or double-stranded breaks in helix to add or remove supercoils
Fluoroquinolones action
Inhibit DNA gyrase (prok. topoisomerase II)
Primase
RNA primer for DNA pol III initiation
DNA pol III
Prok. only, 5’-3’ replication, 3’-5’ exonuclease activity (proofreading). On lagging strand, reads until it gets to primer
DNA pol I
Prok. only. Replaces RNA primer with DNA. 5’-3’ exonuclease activity
DNA ligase
Joins Okazaki fragments
Telomerase
RNA-dependent DNA polymerase that adds DNA to 3’ ends of chromosomes to avoid loss of genetic material with every duplication.
HGPRT role
Recycling back to nucleic acids. Guanine to GMP, Hypoxanthine to IMP (moves away from xanthine and uric acid!!)
APRT role
It is the HGPRT for Adenine. Adenine to AMP.
What is the order of severity of mutations to the genetic code
silent«frameshift.
Transversions (purine to pyrimidine) is worse than transitions (purine to purine)
Most silent mutations found where in codon
In the 3rd position (wobble!)
Sickle cell caused by what mutation
Missense
How to get a frameshift
Delete or add nucleotides not a multiple of 3.
What mutation is duchenne’s
Frameshift
I have a bulky helix-distorting DNA lesion, what do I use?
Nucleotide excision repair. Removes an oligonucleotide containing the damage then DNA pol and ligase fills it in. Pyrimidine dimers and bulky chemical adducts.
I have an altered Base
Base excision repair
Base excision repair:
Base-specific glycosylase recognizes altered base and creates AP site (apurinic,apyrimidinic). One or more nucleotides are removed by AP-endonuclease, which cleaves the 5’ end. Lyase cleaves the 3’ end. DNA pol-beta fills the gap and DNA ligase seals it.
AP-endonuclease action
Forms a single strand break. DNA glycosylase just removes the base by cleaving the N-glycosidic bond. AP endonuclease cleaves the 5’ end of the AP site
Lyase action
Cleaves 3’ end of AP site
Mismatch repair vs. Nucleotide exicision repair
Nucleotide excision for bulky adducts or major distortions to the DNA helix.
Mismatch repair
Repairs errors that occur during DNA synthesis. Usually just transitional errors (laying a C instead of T)
Xeroderma pigmentosum problem
Nucleotide excision repair, prevents repair of pyrimidine dimers because of UV exposure
Spontaneous/toxic deamination repair
Base excision repair. It’s the reason why DNA has thymine, because when it deaminates it turns methylated cytosine which is recognizable. Not cytosine if we had uracil instead.
Hereditary nonpolyposis colorectal cancer problem
Mismatch repair
Ataxia telangiectasia problem
Nonhomologous end joining
Nonhomologous end joining problem
Repairs double stranded breaks. No requirement for homology.
Energy for DNA/RNA production
5’ end of incoming nucleotide bears the triphosphate
Protein synthesis direction
N-terminus to C-terminus
mRNA read
5’ to 3’
Phosphate bond reaction
Triphosphate bond targeted by the 3’ hydroxyl attack.
How to block DNA replication
Modified 3’ OH, preventing addition of the next nucleotide (chain termination)
mRNA start codons
AUG (rarely GUG):
AUG codes for
Euk. methionine which may be removed before translation ends. Prok. formylmethionine (f-met.)
mRNA stop codons
UAA, UGA, UAG (u are annoying, u go away, u are gone)
Promoter regions
TATA boxes and CAAT boxes (weak bonds, easy to open)
Enhancers bind
Transcription factors, may be found in introns
Silencers bind
Repressors, may be found far away, close to, or in an intron, like enhancers.
Most numerous RNA
rRNA (in ribosomes)
Largest RNA
mRNA
Smallest RNA
tRNA
RNA pol I
rRNA
RNA pol II
mRNA
RNA pol III
tRNA
Euk. or prok. have 3 RNA pol
Euk. have 3 RNA pol, prok. have just 1.
alpha-amanitin path
inhibits RNA pol II, severe hepatotoxicity, found in Amanita phalloides (death cap mushrooms)
Initial mRNA from transcription is called
Heterogenous nuclear RNA (hnRNA)
mRNA processing
- 5’ cap (7-methylguanosine cap)
- Polyadenylation at 3’ end (around 200 A’s)
- Splicing out introns
mRNA quality control
Cytoplasmic P-bodies, contain exonucleases, decapping enzymes, and microRNAs; mRNAs may be stored here for future translation
What are P-bodies?
Processing bodies. Decaps and degrades unwanted mRNAs. Stores mRNA for later translation. Aids in translation repression with miRNAs (like siRNAs)
Poly-A polymerase template
No template needed
Polyadenylation signal
AAUAAA
Splicing
- Primary transcript combines with small nuclear ribonucleoproteins (snRNPs) and other proteins to form spliceosome. 2. Lariat-shaped intermediate is generated 3. Lariat is released to precisely remove intron
Splicing mechanistically
A 3’ OH is formed during lariat formation which then allows for an attack at the phosphodiester bond at the 2nd exon leading to splicing out the intron.
anti-smith antibodies
Antibodies to spliceosomal snRNPs (anti-Smith antibodies). Highly specific for SLE.
Anti-U1 RNP antibodies
Highly associated with MCTD
Draw out splicing reaction
….
Abnormal splicing can cause what
Oncogenesis, Beta-thal
Exons vs. introns
Exons are coding
tRNA structure
75-90 nucleotides. Cloverleaf. CCA at 3’ end which binds the amino acid. Anticodon end is opposite 3’ aminoacyl end. The A in CCA binds the aminoacid.
T-arm of tRNA
Contains TPsyC (thymine, pseudouridine, cytosine) sequence necessary for tRNA-ribosome binding.
D-arm of tRNA
Contains dihydrouracil residues necessary for tRNA recognition by the correct aminoacyl-tRNA synthetase.
Acceptor stem of tRNA
The 3’ CCA is the amino acid acceptor site
tRNA charging
Aminoacyl-tRNA synthetase checks AA before and after binding to tRNA, if incorrect, it hydrolyzes it because you can’t fix it afterwards.
How many amino acids for each aminoacyl-tRNA synthetase
One synthetase for every AA
Aminoacyl-tRNA synthesis reaction energy
ATP used to make the bond, but the new bond is used to form the peptide bond.
Anticodon for start codon
UAC (binding to AUG)
tRNA wobble
Only first 2 nucleotide positions of an mRNA codon matter
Initiation of translation
GTP hydrolysis; initiation factors assemble 40S with initiator tRNA and are released when the mRNA and 60S assemble with the complex
Euk. ribosome
40S + 60S = 80S (Even (euk.))
Prok. ribosome
30S + 50S = 70S (Odd (prOk.))
ATP-tRNA
Activation (charging)
GTP-tRNA
Gripping and Going places (translocation)
Initiator methionine binds where
P site
Steps in translation
Aminoacyl-tRNA binds to A site, peptide bond forms, translocation 3 nucleotides over and repeat.
Stop codon reached, then what
Release factor comes in and releases the polypetide
A, P, and E sites
Aminoacyl, peptide, and exit.
Posttranslational modifications
Cleaving N- or C-terminus of zymogen. Phosphorylation, glycosylation, hydroxylation, methylation, acetylation, and ubiquitination.
What are heat shock proteins
e.g. Hsp60, in yeast, are chaperonins expressed at high temps to prevent protein denaturing/misfolding.
Chaperone proteins
Can facilitating or maintain protein folding.
What regulates cell cycle phases
Cyclins, cyclin-dependent kinseases (CDKs), and tumor suppressors.
Which phases are variable in time
G1 and G0. Not G2, when that begins, there is a set time for when it must go to mitosis.
Order of the phases
G1/G0 to S phase to G2 to Mitosis.
What regulates G1 to S phase progression.
Rb, p53
CDKs action
Constitutive and inactive…????
Cyclins
Regulatory proteins that control cell cycle events; phase specfic; activate CDKs
Cyclic-CDK complexes
Must be both activated and inactivated for cell cycle to progress
p53 and Rb
Hypophophorylated Rb and p53 normally inhibit G1-to-S progression
What happens if you mutate p53 or Rb
Unrestrained cell division (e.g. Li-Fraumeni)
What is interphase
G1, S, and G2
What cell lines are G0
Neurons, skeletal and cardiac muscle, RBCs. These are permanent
What cells go from G0 to G1
Hepatocytes, lymphocytes. These are quiescent.
What cells never go to G0
Bone marrow, gut epithelium, skin, hair follices, germ cells
Rough ER purpose
Site of synthesis of secretory (exported) proteins and of N-linked oligosaccharide addition to many proteins
Example of cells with lots of RER
Mucus-secreting goblet cells of the small intestine and antibody-secreting plasma cells
Nissl bodies
RER in neurons, synthesize peptide neurotransmitters for secretion
What do free ribosomes do
Site of synthesis of cytosolic and organella proteins
Smooth ER purpose
Steroid synthesis and detox of drugs and poisons.
Cells with lots of SER
Liver hepatocytes and steroid hormone-producing cells of the adrenal cortex and gonads
Golgi purpose
Movies proteins and lipids from the ER to vesicles and plasma membrane.
Example of specific effects of Golgi
Modifies N-oligosaccharides on asparagine.
Adds O-oligosacchardies on serine and threonine.
Adds mannose-6-phophate to proteins for trafficking to lysosomes.
Mannose-6-phosphate
…
Endosomes purpose
Take stuff from outside the cell or from the Golgi, sending it to lysosomes for destruction or back to the membrane/Golgi for further use.
Inclusion cell disease (I-cell disease) path
Inherited lysosomal storage disorder; defect in phosphotransferase. Golgi can’t phosphorylate mannose residues (i.e. dec. mannose-6-phosphate) on glycoproteins leading to extracellular excretion and not delivered to lysosomes.
I-cell disease presentation
Coarse facial features, clouded corneas, restricted joint movement, and high plasma levels of lysosomal enzymes. Often fatal in childhood.
Signal recognition particle (SRP)
Abundant, cytosolic ribonucleoprotein that traffics proteins from the ribosome to the RER. Absent or dysfunctional SRP leads to proteins accumulating in the cytosol.
Vesicular trafficking proteins
COPI, COPII, and Clathrin.
COPI functions
Golgi to Golgi (retrograde); Golgi to ER
COPII functions
Golgi to Golgi (anterograde); ER to Golgi
Clathrin functions
trans-Golgi to lysosomes; plasma membrane to endosomes (receptor mediated endocytosis (LDL receptor))
Peroxisome
Catabolism of very-long-chain fatty acids, branched-chain fatty acids, and amino acids
Proteasome
Breaks down damaged or ubiquitin tagged proteins. Defects in teh ubiquitin-proteasome system have been implicated in some cases of Parkinson’s.
Microtubule structure
Helical cylinder of polymerized heterodimers of alpha and beta-tubulin. Each dimer uses 2 GTP.
Microtubule location
Flagella, cilia, mitotic spindles
Microtubule growth
Grow slowly (at positive end), collapse quickly
Microtubule in neurons
Slow axoplasmic transport
Molecular motor proteins
Dynein (retrograde to microtubule (+ to -)
Kinesin (anterograde to microtubule (- to +)
Drugs acting on Microtubules
Microtubules Get Poorly Very Poorly: Mebendazole, Griseofulvin, Colchicine, Vincristine/Vinblastine, Paclitaxel
Cilia structure
9+2 microtubule pair arragement with dynein ATPase linking peripheral 9 doublets
Kartageners syndrome presentation
Primary ciliary dyskinesia. Male and female infertility from immotile sperm and dysfunctional fallopian tube cilia. Increased risk of ectopic. Can cause bronchiectasis, recurrent sinusitus, and situs inversus.
Actin and Myosin found in
Muscles, microvilli, cytokinesis, adherens junctions.
Myosin structure
Dimeric, ATP driven motors
Intermediate filaments examples
Used for structure, vimentin, desmin, cytokeratin, lamins, glial fibrillary acid proteins (GFAP), neurofilaments.
Fungal membranes contain what
Ergosterol
What tissue stains vimentin
Connection
Tissue stain desmin
Muscle (desMin)
Tissue stain Cytokeratin
Epithelial Cells
GFAP tissue
NeuroGlia
Neurofilaments tissue
Neurons
Na/K ATPase
3 Na out, 2 K in. Net charge of 1 + out.
Toxins on Na/K ATPase
Ouabain Cardiac glycosides (digoxin and digitoxin)
Cardiac glycosides action
Directly inhibit the Na/K ATPase, which leads to indirect inhibition of Na/Ca exchange leading to increased intracellular calcium and increased cardiac contractility. Too much sodium in cell prevents movement of calcium into outside.
Oubain MOA
Inhibits K+ binding
ATP at what site of Na/K ATPase
Intracellular, fires when the sodium is released (first step). K+ comes in last
Which cartilage is the most common
Type I: 90%
Where do you find Type I, II, III, and IV cartilages (The Important Sites)
I: Bone
II: Cartilage (cartwolage)
III: Blood vessels (big one)
IV: Under the floor (four/basement membrane)
Type I cartialge
Bone (osteoblasts), skin, tendon, dentin, fascia, cornea, late wound repair.
Type II cartilage
Cartilage (including hyaline), vitreous body, nucleus pulposus.
Type III cartilage
Reticulin: Skin, blood vessels, uterus, fetal tissue, granulation tissue
Type IV cartilage
Basement membrane, basal lamina, lens
Alport syndrome path
Defective Type IV collagen
Goodpasture path
Autoantibodies to Type IV
Vascular type of Ehlers-Danlos syndrome)
Type III; uncommon type!
Mnemonic for cartilage
Be So Totally Cool, Read Books.
BST C R B
Osteogenesis imperfecta type I
Type I cartilage decreased production
Collagen synthesis steps
- Synthesis 2. Hydroxylation 3. Glycosylation 4. Exocyotosis 5. Proteolytic processing 6. Cross-linking
- Collagen Synthesis step
Translation of collagen alpha chains (preprocollagen): Gly-X-Y (X and Y are proline or lysine)
Amino acids that make up collagen
Glycine, proline, and lysine
- Collagen hydroxylation step
Hydroxylation of specific proline and lysine residues requiring vit C
- Collagen glycosylation step
Glycosylation of pro-alpha-chain hydroxyline residues and formation of procollagen via hydrogen and disulfide bonds (triple helix of 3 collagen alpha chains). Then leads to 4. Exocytosis
Osteogenesis imperfecta path
Can’t form triple helix of procollagen.
What steps happen in the RER
Synthesis, hydroxylation, and glycosylation.
Look at a figure for Collagen synthesis steps
….
- Proteolytic processing of collagen
Cleavage of disulfide-rich terminal regions of procollagen, transforming it into insoluble tropocollagen.
- Cross linking of collagen
Staggered tropocollagen molecules reinforced by covalent lysine-hydroxylysine cross-linkage (by Cu2+-containing lysyl oxidsae) to make collagen fibrils.
Ehlers-Danlos path
You can’t cross link collagen properly.
Osteogenesis imperfecta presentation
Brittle bone disease. Multiple fractures from minimal trauma. Blue sclerae because of translucency over the choidal veins. hearing loss (abnormal ossicles). Dental imperfections due to lack of dentin. May be confused with child abuse.
Osteo. Imp. genetics
Most common form is Aut. dom. with decreased production of otherwise normal type I collagen.
Ehlers-Danlos presentation
Hyperextensible skin, tendency to bleed (easy bruising), and hypermobile joints. May be associated with joint dislocation, berry and aortic aneurysms, and organ rupture.
Ehlers-Danlos types
6+ types. May be aut. dom. or rec. Hypermobility type: Most COMMON. Classical type (joint and skin symptoms): Mutation in type V collagen. Vascular type (vascular and organ rupture): Deficient type III collagen
Menkes disease
Connective tissue disease caused by impaired copper absorption and transport. Leads to decreased activity of lysyl oxidase (copper is a necessary cofactor). Results in brittle, kinky hair, growth retardation and hypotonia.
Elastin is found where
Skin, lungs, large arteries, elastic ligaments, vocal cords, ligamenta flava
Elastin structure
Rich in proline and glycine, nonhydroxylated forms. ????
Tropoelastin with fibrillin scaffolding?
….??
Elastin cross-linking
Takes place extracelluarly and gives elastin its elastic properties
What breaks down and prevents breakdown of elastin?
Elastase breaks down, inhibited by alpha1-antitrypsin.
Marfan syndrome
Defect in fibrillin, a glycoprotein that forms a sheath around elastin
Emphysema
Can be caused by alpha1-antitrypsin deficiency
Wrinkles of aging caused by
Lower collagen and elastin production
Diagnosing neonatal HIV or herpes encephalitis
PCR
Southern blot steps
DNA electrophoresed on gel then transferred to filter. Then denatured and exposed to radiolabeled DNA probe.
Northen blot
RNA
Western blot
Protein with antibody probe
Confirmatory test for HIV
Western blot after + ELISA
Southwestern Blot
DNA-binding proteins (Transcription factors) using labeled oligonucleotide probes
Microarrays benefits
Can profile gene expression levels of thousands of genes simultaneously to study diseases and treatments. Can detect SNPs and copy number variations (CNVs) for genotyping, clinical genetic testing, forensic analysis, cancer mutations, and genetic linkage analysis.
