Bio Chemisity Flashcards
Chromatin structure
Chromatin structure
DNA exists in the condensed, chromatin form in order to fit into the nucleus. Negatively charged DNA loops twice around positively charged histone octamer to form nucleosome “bead.” Histones are rich in the amino acids lysine and arginine. H1 ties nucleosome beads together in a string.
In mitosis, DNA condenses to form chromosomes.
Think of “ beads on a string.”
H1 is the only histone that is not in the nucleosome core.
Heterochromatin
Heterochromatin
Condensed, transcriptionally inactive, sterically inaccessible.
HeteroChromatin = Highly Condensed.
Euchromatin
Euchromatin
Less condensed, transcriptionally active, sterically accessible.
Eu = true, so “truly transcribed.”
DNA methylation
DNA methylation
Template strand cytosine and adenine are methylated in DNA replication, which allows mismatch repair enzymes to distinguish between old and new strands in prokaryotes.
Histone methylation
Histone methylation
Inactivates transcription of DNA.
Methylation makes DNA Mute.
Histone acetylation
Histone acetylation
Relaxes DNA coiling, allowing for transcription
Acetylation makes DNA Active.
Nucleotides
Nucleotides
PURines (A, G)-2 rings. G has a double bond Oxygen. Think go go go G (Guanine) has an O
PYrimidines (C, U, T)-1 ring. Think CUT the pie (has one ring/circle
Guanine has a ketone. Thymine has a methyl.
Deamination of cytosine makes uracil.
Uracil found in RNA; thymine in DNA.
G-C bond (3 H bonds) stronger than A-T bond (2 H bonds).
higher amount of G-C content equals higher melting point.
- *PUR**e As Gold.
- *CUT** the PYrimidines (pie). Thymine has a methyl.
GAG-Amino acids necessary for purine synthesis :
Glycine
Aspartate
Glutamine
NucleoSide= base + ribose (Sugar).
NucleoTide= base + ribose + phosphaTe;
linked by 3’-5’ phosphodiester bond.
De novo purine synthesis
Purines
Start with sugar + phosphate (PRPP)
Add base
De novo pyrimidine synthesis
Pyrimidines
Make temporary base (orotic acid)
Add sugar + phosphate (PRPP)
Modify base
Ribonucleotides are synthesized first and are converted to deoxyribonucleotides by ribonucleotide reductase.
Carbamoyl phosphate is involved in 2 metabolic pathways: de novo pyrimidine synthesis and the urea cycle. Ornithine transcarbamoylase deficiency (OTC, key enzyme in the urea cycle) leads to an accumulation of carbamoyl phosphate, which is then converted to orotic acid.
Various antineoplastic and antibiotic drugs function by interfering with purine synthesis:
- Hydroxyurea inhibits ribonucleotide reductase
- 6-mercaptopurine (6-MP) blocks de novo purine synthesis
- 5-Auorouracil ( 5-FU) inhibits thymidylate synthase (decreased deoxythymidine monophosphate [dTMP])
- Methotrexate (MTX) inhibits dihydrofolate reductase decreased dTMP)
- Trimethoprim (TMP) inhibits bacterial dihydrofolate reductase (decreased dTMP)
Orotic aciduria
Orotic aciduria
Inability to convert orotic acid to UMP (de novo pyrimidine synthesis pathway) because ofdefect in UMP synthase (a bifunctional enzyme). Autosomal recessive.
FINDINGS: increased orotic acid in urine, megaloblastic anemia (does not improve with administration ofvitamin B12 or folic acid), failure to thrive. No hyperammonemia (vs. OTC deficiency- increased orotic acid with hyperammonemia).
TREATMENT: Oral uridine administration.
Purine salvage deficiencies
Purine salvage deficiencies
1) Adenosine deaminase deficiency: Excess ATP and dATP imbalances nucleotide pool via feedback inhibition of ribonucleotide reductase -> prevents DNA synthesis and thus decrease lymphocyte count. One of the major causes of SCID. Autosomal recessive.
Severe Combined Immunodeficiency Disease (SCID) happens to kids.
1st disease to be treated by experimental human gene therapy.
2) Lesch-Nyhan syndrome: Defective purine salvage owing to absence of HGPRT, which converts hypoxanthine to IMP and guanine to GMP. Results in excess uric acid production and de novo purine synthesis. X-linked recessive.
Findings: retardation, self-mutilation, aggression, hyperuricemia, gout, choreoathetosis.
learning aid: He’s Got Purine Recovery Trouble.
Genetic code features
Genetic code features
Unambiguous: Each codon specifies only l amino acid.
Degenerate/ redundant: Most amino acids are coded by multiple codons.
- Exceptions: methionine and tryptophan encoded by only l codon (AUG and UGG, respectively).
Commaless, nonoverlapping: Read from a fixed starting point as a continuous sequence of bases.
- Exceptions: some viruses.
Universal: Genetic code is conserved throughout evolution.
- Exception in humans: mitochondria.
Point mutations in DNA
Point mutations in DNA
Severity of damage: silent< missense< nonsense< frameshift.
Silent: Same amino acid, often base change in 3rd position of codon (tRNA wobble).
Missense: Changed amino acid (conservative-new amino acid is similar in chemical structure).
Nonsense: Change resulting in early stop codon.
- learning aid: Stop the nonsense!
Frameshift: Change resulting in misreading of all nucleotides downstream, usually resulting in a truncated, nonfunctional protein.
DNA replication
DNA replication
In both prokaryotes and eukaryotes, DNA replication is semiconservative and involves both continuous and discontinuous (Okazaki fragment) synthesis.
Origin of replication
Origin of replication
Particular consensus sequence ofbase pairs in genome where DNA replication begins. May be single (prokaryotes) or multiple (eukaryotes).
DNA Replication fork
DNA Replication fork
Y-shapecl region along DNA template where leading and lagging strands are synthesized.
Helicase
Helicase unwinds DNA template at replication fork.
Single-stranded binding proteins
Single-stranded binding proteins prevent strands from reannealing.
DNA topoisomerases
DNA topoisomerases
Create a nick in the helix to relieve supercoils created during replication.
- Fluoroquinolones-inhibit DNA gyrase (prokaryotic topoisomerase II).
Primase
Primase makes an RNA primer on which DNA polymerase III can initiate replication.
DNA polymerase Ill
DNA polymerase Ill
Prokaryotic only. Elongates leading strand
by adding cleoxynucleoticles to the 3’ encl. Elongates lagging strand until it reaches primer of preceding fragment. 3’ -> 5’ exonuclease activity “proofreads” each aclclecl nucleotide.
- DNA polymerase III has 5’ -+ 3’ synthesis and proofreads with 3’ -+ 5’ exonuclease.
DNA polymerase I
DNA polymerase I
Prokaryotic only. Degrades RNA primer; replaces it with DNA.
- Has same functions as DNA polymerase III but also excises RNA primer with 5’ -+ 3’ exonuclease.
DNA ligase
DNA ligase
Catalyzes the formation ofphosphodiesterase bond within a strand of double-stranded DNA (i.e., joins Okazaki fragments).
- Seals.
Telomerase
Telomerase enzyme adds DNA to 3’ ends of chromosomes to avoid loss of genetic material with every duplication.
Types of RNA
Types of RNA
rRNA is the most abundant type.
mRNA is the longest type.
tRNA is the smallest type.
memory aid: Rampant, Massive, Tiny.
Start and stop codons
Start and stop codons
mRNA start codons: AUG (or rarely GUG).
- memory aid: AUG inAUGurates protein synthesis.
Eukaryotes: Codes for methionine, which may be removed before translation is completed.
Prokaryotes: Codes for formylmethionine (f-met).
mRNA stop codons: UAA, UAG, UGA.
- memory aid:
- U Are Awesome
- U Are Great
- U Gonna get an A!
or
- memory aid:
- UGA = U Go Away.
- UAA = U Are Away.
- UAG= U Are Gone.
Fundional organization of the gene
Fundional organization of the gene
Promoter
Promoter
Site where RNA polymerase and multiple other transcription factors bind to DNA upstream from gene locus (Kf-rich upstream sequence with TKfA and CAAT boxes).
- Promoter mutation commonly results in dramatic decrease in amount of gene transcribed.
Enhancer
Enhancer
Stretch of DNA that alters gene expression by binding transcription factors.
- Enhancers and silencers may be located close to, far from, or even within (in an intron) the gene
Silencer
Silencer
Site where negative regulators (repressors) bind.
- whose expression it regulates.
Eukaryotes RNA polymerases
Eukaryotes RNA polymerases
RNA polymerase I makes rRNA (most numerous RNA, rampant).
RNA polymerase II makes mRNA (largest RNA, massive).
RNA polymerase III makes tR  A (smallest RNA, tiny).
No proofreading function, but can initiate chains. RNA polymerase II opens DNA at promotor site
- I, II, and III are numbered as their products are used in protein synthesis.
- alpha-amanitin, found in Amanita phalloides (death cap mushrooms), inhibits RNA polymerase II. Causes severe hepatotoxicity ifingested.
Prokaryotes Polymerases
Prokaryotes Polymerases
l RNA polymerase (multisubunit complex) makes all 3 kinds of RNA.
RNA processing (eukaryotes)
RNA processing (eukaryotes)
Initial transcript is called heterogeneous nuclear RNA (hnRNA). hnRNA destined for translation is called pre-mRA.
- Only processed RNA is transported out of the nucleus.
Processing occurs in nucleus. After transcription:
- Capping on 5’ end (addition of 7-methylguanosine cap)
- Polyadenylation on 3’ end (“” 200 1\s)
- Poly-A polymerase does not require a template.
- AAUAAA =polyadenylation signal.
- Splicing out of introns
- Capped, tailed, and spliced transcript is called mRNA.
Splicing of pre-mRNA
Splicing of pre-mRNA
1) Primary transcript combines with snRNPs and other proteins to form spliceosome.
2) Lariat-shaped (looped) intermediate is generated.
3) Lariat is released to remove intron precisely and join 2 exons.
Patients with lupus make antibodies to spliceosomal snRNPs.
lntrons vs. exons
lntrons vs. exons
Exons contain the actual genetic information coding for protein.
- Different exons can be combined by alternative splicing to make unique proteins in different tissues (e.g., beta-thalassemia mutations).
lntrons are intervening noncoding segments of DNA.
learning aid: lntrons are intervening sequences and stay in the nucleus, whereas exons exit and are expressed.
tRNA Structure
tRNA Structure
75-90 nucleotides, secondary structure, cloverleafform, anticodon end is opposite 3’ aminoacyl end. All tRNAs, both eukaryotic and prokaryotic, have CCA at 3’ end along with a high percentage of chemically modified bases. The amino acid is covalently bound to the 3’ end of the tRNA.
Memory aid: CCA: Can Carry Amino acids.
tRNA Charging
tRNA Charging
Aminoacyl-tRNA synthetase (1 per amino
acid, “matchmaker,” uses ATP) scrutinizes amino acid before and after it binds to tRNA. If incorrect, bond is hydrolyzed. The amino acid-tRNA bond has energy for formation of peptide bond. A mischarged tRNA reads usual codon but inserts wrong amino acid.
Aminoacyl-tRNA synthetase and binding of charged tRNA to the codon are responsible for accuracy of amino acid selection.
Tetracyclines bind 30S subunit, preventing attachment of aminoacyl-tRNA.
tRNA wobble
tRNA wobble
Accurate base pairing is required only in the first 2 nucleotide positions of an mRNA codon, so codons differing in the 3rd “wobble” position may code for the same tRNA/amino acid (as a result of degeneracy of genetic code).
Initiation of protein synthesis
Initiation of protein synthesis
Activated by GTP hydrolysis, initiation factors (eukaryotic IFs) help assemble the 40S ribosomal subunit with the initiator tRNA and are released when the mRNA and the ribosomal subunit assemble with the complex.
Eukaryotes: 40S + 60S -+ 80S (Even).
PrOkaryotes: 30S + 50S – 70S (Odd).
ATP-tRNA Activation (charging).
- *G**TP-tRNA Gripping and Going places
(translocation) .
Elongation
Elongation
1) Aminoacyl-tRNA binds to A site (except for initiator methionine)
2) Ribosomal rRNA (“ribozyme”) catalyzes peptide bond formation, transfers growing polypeptide to amino acid in A site.
3) Ribosome advances 3 nucleotides toward 3’ end of mRNA, moving peptidyl tRNA to P site (translocation)
Learning aid: Think of “going APE”:
A site = incoming Aminoacyl-tRNA.
P site = accommodates growing Peptide.
E site = holds Empty tRNA as it Exits.
Termination
Termination
Stop codon is recognized by release factor, and completed protein is released from ribosome.
Antibiotics that act as protein synthesis inhibitors:
Antibiotics that act as protein synthesis inhibitors:
• Aminoglycosides bind 30S and inhibit
formation of initiation complex and cause
misreading of mRNA
- Tetracyclines bind 30S and block aminoacyl tRNA from entering the acceptor site
- Chloramphenicol binds 50S and inhibits peptidyl transferase
- Macroslides bind 50S and prevent release of uncharged tRNA after it has donated its amino acid
Learning aid: The exam you took to apply to med school Macroslides Choramphenicol Aminoglycosides Tetracycline
Trimming (Posttranslational modifications)
Trimming (Posttranslational modifications)
Removal of N- or C-terminal propeptides from zymogens to generate mature proteins.
Covalent alterations (Posttranslational modifications)
Covalent alterations (Posttranslational modifications)
Phosphorylation, glycosylation, hydroxylation, methylation, and acetylation.
Learning aid: please give him my address!
Proteasomal degradation
(Posttranslational modifications)
Proteasomal degradation (Posttranslational modifications)
Attachment of ubiquitin to defective proteins to tag them for breakdown.
Cell cycle phases
Cell cycle phases
Checkpoints control transitions between phases of cell cycle. This process is regulated by cyclins, CDKs, and tumor suppressors. Mitosis (shortest phase): prophase-metaphase-anaphase-telophase. G1 and G0 are of variable duration.
CDKs
CDKs
Cyclin-dependent kinases; constitutive and inactive.
Cyclins
Cyclins
Regulatory proteins that control cell cycle
events; phase specific; activate CDKs.
Tumor suppressors (cell cycle)
Tumor suppressors (cell cycle)
p53 and hypophosphorylated Rb normally inhibit G1-to-S progression; mutations in these genes result in unrestrained cell division.
Permanent (cell type)
Permanent (cell type)
Remain in G0, regenerate from stem cells.
- Neurons, skeletal and cardiac muscle, RBCs.
Cell trafficking
Cell trafficking
Golgi is the distribution center for proteins and lipids from the ER to the vesicles and plasma membrane.
- It modifies N-oligosaccharides on asparagine.
- It adds O-oligosaccharides on serine and threonine.
-
It adds mannose-6-phosphate to proteins for trafficking to lysosomes.
- 1-cell disease (inclusion cell disease)-inherited lysosomal storage disorder; failure of addition of mannose-6-phosphate to lysosome proteins (enzymes are secreted outside the cell instead of being targeted to the lysosome). Results in coarse facial features, clouded corneas, restricted joint movement, and high plasma levels of lysosomal enzymes. Often fatal in childhood.
Endosomes are sorting centers for material from outside the cell or from the Golgi, sending it to lysosomes for destruction or back to the membrane/Golgi for further use.
Vesicular trafficking proteins
- COPI: Golgi -> Golgi (retrograde); Golgi -> ER.
- COPII: Golgi -> Golgi (anterograde); ER -> Golgi.
- Clathrin: trans-Golgi -> lysosomes; plasma membrane-> endosomes (receptor-mediated endocytosis.
Microtubule
Microtubule
- Cylindrical structure composed of a helical array of polymerized dimers of alpha- and beta-tubulin.
- Each dimer has 2 GTP bound.
- Incorporated into flagella, cilia, mitotic spindles.
- Grows slowly, collapses quickly.
- Also involved in slow axoplasmic transport in neurons.
Microtubule molecular motor proteins
Microtubule molecular motor proteins
Molecular motor proteins is the transport cellular cargo toward opposite ends of microtubule tracks.
- Dynein= retrograde to microtubule (+ to -)
- Kinesin =anterograde to microtubule (- to +)
Cilia structure
Cilia structure
9 + 2 arrangement of microtubules.
Axonemal dynein-ATPase that links peripheral 9 doublets and causes bending of cilium by differential sliding of doublets.
- Kartagener’s syndrome (primary ciliary dyskinesia) - immotile cilia clue to a dynein arm defect. Results in male infertility (immotile sperm) and decreased female fertility, bronchiectasis (abnormal widening of the bronchi or its branches leading to increased risk of respiratory infection), and recurrent sinusitis (bacteria and particles not pushed out); associated with situs inversus.
sodium-potassium pump
sodium-potassium pump
Na+-K+ ATPase is located in the plasma membrane with ATP site on cytosolic side. For each ATP consumed, 3 Na+ go out and 2 K+ come in. During cycle, pump is phosphorylated.
Ouabain inhibits by binding to K+ site. Cardiac glycosides (digoxin and digitoxin) directly inhibit the Na+-K+ ATPase, this leads to indirect inhibition of Na+/ Ca2+ exchange, which leads to increased [Ca2+]i -which leads to increased cardiac contractility.
collagen synthesis inside fibroblasts/ outside fibroblasts
collagen synthesis inside fibroblasts
1) Synthesis (RER): Translation of collagen a chains (preprocollagen) -usually Gly-X-Y (X and Y are proline or lysine).
2) Hydroxylation (ER): Hydroxylation ofspecific proline and lysine residues (requires vitamin C; deficiency leads to scurvy).
3) Glycosylation (ER): Glycosylation of pro-alpha-chain hyclroxylysine residues and formation of procollagen via hydrogen and disulfide bonds (triple helix of 3 collagen a chains). Problems forming triple helix leads to osteogenesis imperfecta.
4) Exocytosis: Exocytosis of procollagen into extracellular space.
collagen synthesis outside fibroblasts
5) Proteolytic processing: Cleavage of disulfide-rich terminal regions of procollagen, transforming it into insoluble tropocollagen.
6) Cross-linking: Reinforcement of many staggered tropocollagen molecules by covalent lysine-hydroxylysine cross-linkage (by Cu2+-containing lysyl oxidase) to make collagen fibrils. Problems with cross-linking leads to Ehlers-Danlos.
Osteogenesis imperfecta
Osteogenesis imperfecta- (collagen disease)
Genetic bone disorder (brittle bone disease) caused by a variety of gene defects.
Most common form is autosomal dominant with abnormal type I collagen (Most common (90%)-Bone, Skin, Tendon, dentin, fascia, cornea, late wound repair.), causing:
- Multiple fractures with minimal trauma;
- may occur during the birth process
- Blue sclera due to the translucency of the connective tissue over the choroidal veins
- Hearing loss (abnormal middle ear bones)
- Dental imperfections clue to lack of dentin
May be confused with child abuse. Incidence is 1 : 10,000.
Hardy-Weinberg population genetics
Hardy-Weinberg population genetics
If a population is in Hardy-Weinberg equilibrium and if p and q are the frequencies of separate alleles, then: p2 + 2pq + q2 = 1 and p + q = 1, which implies that:
p2 = frequency of homozygosity for allele p q2 = frequency of homozygosity for allele q 2pq = frequency of heterozygosity (carrier frequency, if an autosomal recessive disease). The frequency of an X-linked recessive disease in males = q and in females = q2.
Example: Hardy-Weinberg law assumptions include:
- No mutation occurring at the locus
- Natural selection is not occurring
- Completely random mating
- No net migration
Autosomal dominant
Autosomal dominant
Often due to defects in structural genes. Many generations, both male and female, affected.
Often pleiotropic (one gene effecting multiple seemingly unrelated phenotypes). Family history crucial to diagnosis.
Legend:
purple= uninfected
pink= infected
Autosomal recessive
Autosomal recessive
25% of offspring from 2 carrier parents are affected. Often due to enzyme deficiencies. Usually seen in only 1 generation.
Commonly more severe than dominant disorders; patients often present in childhood. Increased risk in consanguineous families.
X-linked recessive
X-linked recessive
Sons of heterozygous mothers have a 50% chance of being affected. No male-to-male transmission.
Commonly more severe in males. Females usually must be homozygous to be affected.
X-linked dominant
X-linked dominant
Transmitted through both parents. Mothers transmit to 50% of daughters and sons; fathers transmit to all daughters but no sons.
Hypophosphatemic rickets—formerly known as vitamin D–resistant rickets. Inherited disorder resulting inphosphate wasting at proximal tubule. Results in rickets-like presentation.
Mitochondrial inheritance
Mitochondrial inheritance
Transmitted only through the mother. All offspring of affected females may show signs of disease.
Variable expression in a population or even within a family due to heteroplasmy.
Mitochondrial myopathies—rare disorders; often present with myopathy, lactic acidosis and CNS disease. 2° to failure in oxidative phosphorylation. Muscle biopsy often shows “ragged red fibers.”
