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LECTURE: COMPOSITION

inorganic composition

molecular species

H, O, C, N, Ca - living organism

O, Si, Ca, Fe, Ca - earth crust

Da = 10^-24 g

virus

protein > DNA > water

bacterial cell

water >( protein > RNA > DNA; 30%)

human fibroblast

water > RNA > protein > DNA

30-50% of genome has no known function

15-20% are unique to each organism

complexity of genome by number of genes? not a good indicator of complexity

molecular weight of cell components

organelle(10^9), macromolecular complexes(10^6 - 10^9), individual macromolecules(10^4 - 10^9), monomeric subunits(10^2 - 10^3), inorganic subunits(< 10^2)

serum composition units and scale? (roughly, biggest component?)

Cations : Na+(135mM) > K+(5 mM) > Ca++(3 mM) > Mg++(1.2 mM)
Anions: Cl-(105mM) > HCO3-(30 mM) > phosphate (1.5mM)
Proteins: total = 80 g/l, albumin (60 g/l), most abundant protein in serum
fuels: glucose (6mM)
hormones (200pM)

BMI = 65 kg/1.73m = 35.8

pico 10^-12

what's the range of light microscope?

- chloroplast to fish eggs

electron microscope?

- small molecules to plant and animal cells

smallest bacteria 0.3 microns

smallest eukaryote 4 microns

largest virus

RNA most reliable for genome complexity

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LECTURE : STRUCTURE OF AMINO ACIDS AND PROTEINS

amino acid = amino group, carboxyl group and R and hydrogen

L vs D isomers - chirality of amino acid

peptide - strand of amino acid linked by peptide bond(formed by condensation)

cofactor - iron, copper, manganese, etc that facilitate enzyme binding, cofactors can be inorganic or organic

Aliphatic, non-polar

Glycine G (Gly)

Alanine A (Ala)

Proline P (Pro)

Valine V (Val)

Leucine L (Leu)

Isoleucine I (Ile)

Methionine M (Met)

Aromatic groups

Phenylalanine F (Phe)

Tyrosine Y (Tyr)

Tryptophan W (Trp)

Polar, uncharged

-OH, -S

Serine S (Ser)

Threonine T (Thr)

Cysteine C (Cys)

1. carboxamide

Asparagine N(Asn)

Glutamine Q (Gln)

Positively charged

Lysine K(Lys)

Arginine R (Arg)

Histidine H (His)

Negatively charged

Aspartate D (Asp)

Glutamate E (Glu)

Angles

peptide bond formed Merrifield synthesis

Almost all peptide bonds in protein are trans, except X-Pro linkage that is cis/trans

peptide bond planar, cannot rotate

C(α) – N => φ angle

C(α) – C => ψ angle

Free to rotate

Conformations of protein can be specified this way

Amino acids can be post-translationally modified

1. cross links (very common)

Cysteine, disulfide residues used to link two polypeptides together, stabilize protein

2. phosphorylation (common in signal transduction)

phosphate group added to Ser, Thr, Tyr, His

3. Glycosylation (targeting, cell-surface display)

4. Methylation (epigenetic code in nucleosome)

Lys have methyl groups attached to them

5. Hydroxylation

Hydroxyl added to proline structure, defect w/o hydroxylation in connective tissue

Common in various types of collagen structure, defect -> loose joints

6. lipidation (targeting, signal transduction)

dipeptide

oligopeptide

protein (~40 a.a.)

enzymes -> ~100 a.a.

primary structure a sequence of R groups

talk about peptide from N-terminus to C-terminus

Alanyl-Glycine (Ala-Gly)

Glycyl-Alanine (Gly-Ala)

N-terminus peptide is translated first and then ends in C-terminal peptide

alanine, glutamic acid, leucine, methionine are preferred in alpha helix, glycine and tyrosine and serine almostn ever found in alpha helix

proline is common beta turns

Sickle cell anemia

Molecular disease - Linus Pauling

Glu (6) – Val (6) in beta chain of Hb ; hydrophilic to hydrophobic; the hydrophobic amino acid may try to aggregate with other hydrophobic molecules around the subunit, causing polymerization -> sickle cell shape

Protein purification

1. Lyse cells

2. Centrifugation

3. Fractionation (salting out)

4. Column chromatography

1. difference in protein charge, size, binding characteristics (affinity, antibodies, tag (His residue at N terminus) bind to nickel, very powerful)

chromatography types - affinity based most commonly used

ion exchange, beads negative, proteins with negative charges elute faster

Gel electrophoresis to check purity

Electric field, protein migrate through gel as a function of molecular weight

SDS denatures protein - disulfide bond breaks?

Sequencing protein

direct sequencing of peptides by chemical methods, difficult, rarely used; N-terminal sequence (Edman degradation), C-terminal (enzyme carboxypeptidase)

DNA sequencing of genes Most common way of obtaining massive amounts of sequence

Mass spectrometry of peptides (powerful) common way to identify particular protein after gel electrophoresis in complex mixtures

Reverse genetics

Protein -> all possible DNA sequences -> isolation of the gene; looking for possible genetic phenotypes from known sequence

Protein structure / folding

Favored by hydrophobic effect, hydrogen bonds, electrostatics

Modulated by side chain/main chain steric interactions

Opposed by loss of entropy

Non-covalent bonds

H-bond

Ionic

Hydrophobic

Van der Waals

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LECTURE : STRUCTURE OF NUCLEIC ACIDS

Chemistry of bases

5. bases are flat rings

6. bases are stacking on each other; they are planar and pi-orbital reinforce the stack

o purine stack better than pyrimidines

7. bases can form hydrogen bonds

o hydrogen bonds essential in maintaining tertiary structures

8. Chargaff’s rule (same amount of G/A and C/T)

9. Bases undergo tautomerism and resonance e.g. cytosine, then it can bind to thymine??

Nucleic acids are negatively charged (phosphate groups)

10. Proteins that bind to them tend to be positively charged

2’ OH group (DNA has H in 2’) makes the RNA more unstable, RNA can be hydrolyzed very easily by base

5' of carbon of sugar is where the phosphate is attached, and 3' is where is the hydroxyl group, 1' is where the base is

nucleoside - no phosphate group

NMP, NDP, NTP

RNA structure and function

11. mRNA(messenger) is one function

12. cellular RNA single stranded

13. many RNA form complex structures

14. many RNAs associated with proteins (RNP), e.g. telomerase, ribozyme

15. RNAs catalytic activity (ribozyme)

16. miRNA play regulatory roles
17. non-coding RNAs, mRNA, tRNA, miRNA present in both prokaryote and eukaryote
    

Enzymatic heart of ribosome is ribozyme (PT catalytic site no green); ribosomes are mainly composed of RNA and little protein

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LECTURE: pH

important properties of water

Kw = 1.0 * 10^-14 = [H+] [OH-], (mol/dm^3)^2

pH = -log[H+]

[H+]=10^-7; pH = 7

[HA] = [H+][A-]

Ka = ([H+][A-])/[HA]

-logKa = pKa

[H+] = Ka*[HA]/[A-]

-Log[H+] = -log(Ka) – log([HA]/[A-])

pH = pKa + log[A-]/[HA]

blood pH slightly alkaline

bigger Ka, stronger acid, lower pKa, stronger acid

when pH = pKa, max buffering capacity

Henderson Hasselbalch equation remember???

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DNA REPLICATION

General properties of DNA replication

17. semi-conservative

18. nucleotide added at 3’ end; DNA polymerase moves from 5’ to 3’ direction

19. requires an RNA primer; primase synthesizes short RNA primer
    

20. semi-discontinuous

o strand that is continuously synthesized in the direction of fork movement referred as leading strand; the other strand is lagging strand template, synthesized in short fragments Okazaki fragment

o asymmetric process: one continuous, one discontinuous

Okazaki fragments are joined by DNA ligase

21. initiation occurs at defined origin of replication

22. replication forks are bidirectional

DNA polymerase

23. cannot start de novo

24. requires an RNA primer, made by primase

Ligase joins Okazaki fragments

25. RNA primer removed, then the nick sealed by DNA ligase

DNA replication enzymology

26. DNA polymerase for leading and lagging strand

27. DNA helicase – separate strands

28. Gap in lagging strand template between Okazaki fragments; single stranded DNA is stabilized by single-stranded binding protein (SSB in prokaryote, RPA-replication protein/factor A, in eukaryote)

29. Sliding clamp: encircles duplex (newly synthesized) DNA, slides along DNA, helps tether DNA polymerase to the template

o Beta-clamp in prokaryote (dimer)

o PCNA in eukaryote (homotrimer), PCNA also serves as landing pad for many DNA repair and checkpoint proteins

30. Clamp loader loads sliding clamp to DNA

31. Topoisomerases break DNA strands, move them around and rejoin them

o Splits a DNA strand and pass the intact strand and then put the split DNA back together

o Also important in transcription(as well as replication)

o Type I breaks one strand(passes the other of sample duplex), type II breaks both strands of duplex

Replisome

- two polymerases interact with each other in some way: DNA polymerases of leading and lagging strands both move together in the same direction; therefore DNA is looped.

- in lagging strand, finish Okazaki fragment, easier to jump to next site of Okazaki fragments

- strand separation by helicase creates supercoiling

Replication from each origin is bidirectional

32. two forks of one double stranded DNA, lagging and leading reversed opposite side

eukaryotic chromosome has multiple origins

33. early origin fires early, late origin fires later

End replication problem

34. lagging strand’s RNA primer removed, then DNA polymerase can’t normally fill this gap; each replication results in lagging strand shorter => replicative senescence
35. leading strand 3'

35. telomeres = specialized structure/sequence at chromosome ends

o highly repetitive (GGGTTG)

36. telomerase – RNA-dependent DNA polymerase that extends lagging strand template

o adds more repeats enough to allow another Okazaki fragment to be added

37. somatic cells don’t use telomerase so they undergo senescence

38. tumor cells have telomerase that immortalizes them

Replication fidelity

39. DNA polymerase 5’ -> 3’: error rate 1/10^5

40. 3’ -> 5’ exonucleolytic proofreading increases fidelity by order of 10^2

41. Strand-directed mismatch repair increases fidelity by order of 10^2

42. => 1 / 10^9 nucleotides polymerized error rate

43. Price of high fidelity is a very slow enzyme

Proofreading - during replication

44. DNA polymerase won’t add to a 3’ end of a nucleotide that is not base-paired with template (RNA polymerase doesn’t have this function)

45. A – C mis-pair not properly base-paired

46. If 3’ end is not properly base-paired, that end will be sent to editing site of DNA polymerase, the false nucleotide clipped off

47. The clipped end goes back to polymerization site to attempt replication again
50. Mismatch repair (MutS and MutL proteins involved) - after replication, but part of proofreading function
    

o Recognize distortion in DNA, usually bound by some protein

o Complex finds a nick(differentiates new from old DNA strand) in new DNA strand, when nick found, the segment cut off and DNA synthesis in that segment is repeated

e.g. hereditary nonpolyposis colorectal cancer(HNPCC) results from defective mismatch repair

Positive and negative supercoil

why do we not have DNA primer in replication?

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DNA repair and recombination

Damage = chemical alteration in DNA

Mutation = permanent change in DNA

Change in a single nucleotide A/T -> G/C

Insertion/deletion of small number of nucleotide => homopolymer runs, same nucleotide over and over e.g. AAAAAA

Chromosome rearrangements

Insertions/deletion, duplication, inversion, translocation(exchange of pieces between non-homologous chromosomes)

Changes in chromosome number (aneuploides)

3 -> trisomy e.g. Down syndrome (trisomy 21)

Two classes of genes that maintain genome stability

Caretakers – repair genes, act directly on DNA

Gatekeeper – control cell cycle e.g. checkpoint proteins

Major sources of mutation

DNA replication errors – e.g by polymerase, correct by proof-reading and post replicative mismatch repair

DNA damage

49. spontaneous: as a result of normal metabolism in cells e.g. oxidative damage

50. induced: come from environment, chemical found in tobacco smoke, UV, ionizing radiation

If there’s damage in DNA,

1. damage pre-replication repair

a. direct reversal, DNA is directly repaired

b. excision repair, damage is excised from DNA

2. replication

c. translesion synthesis – specialized DNA polymerase, large pocket site for repair, downside is that it’s a very sloppy polymerase

d. homologous recombination

3. post-replication

e. mismatch repair

DNA damage

Altering base pairing properties, or destroys ability to base-pair

Common miscoding DNA lesions

1. Cytosine deanimated to uracil; CG -> T(U)A mutations

2. Guanine oxidated by ROS(reactive oxygen species) to 8-oxo guanine which pairs with A or C

Common lesions that block DNA polymerase

1. abasic sites (bond between base and sugar breaks)

2. UV-induced photoproducts (between pyrimidines)

a. Cyclobutyl dimer formed between covalent Ts, prevents DNA from getting into active site of polymerase

b. 6-4 photoproduct, linkage between pyrimidines, also block DNA polymerases

DNA if not repaired, can be bypassed/tolerated

1. homologous recombination

a. undamaged strand of DNA is used as a template to replicate and bypass the lesion; high fidelity

2. translesion synthesis, specialized DNA polymerases that come and fill in directly over the lesion; low fidelity

b. normal T, 75 nucleotide, but if thymine dimer present, stops at 44

c. if polymerase eta used, it is able to go right through the damage => bypass is error free

Repair of damaged DNA

51. direct reversal: e.g. thymine-thymine(pyrimidine) dimer caused by UV can be broken by photolyase + white light, also enzyme alkyl transferase removes alkyl group from base

52. base-excision repair – single base excised

o Glycosylase – each highly specific, recognize specific type of lesion; e.g. UNG – removes uracil, OGG – removes 8-oxoG

§ Cleaves sugar-base linkage, sugar phosphate backbone remains intact

o AP endonuclease(and phosphodiesterase) removes sugar phosphate, change abasic site to single nucleotide gap

o DNA polymerase adds new nucleotides, DNA ligase seals nick

53. nucleotide excision repair – oligonucleotide excised

o e.g. pyrimidine dimer removal

o recognizes any gross/bulky type of helix distorting lesion

o binds to the lesion, makes nicks on the strand that contains lesion on both sides, by nuclease

o DNA helicase removes the nicked strand

o DNA polymerase and ligase fill the gap

o Defective nucleotide excision repair -> e.g. xeroderma pigmentosum; XP can also result from absence of translesion synthesis DNA polymerase pol eta

54. Homologous recombination/Repair of double strand breaks

55. Double strand breaks very detrimental compared to single strand break

56. usually accidental e.g. x-ray UV

57. physiological, intentional – meiosis

o => meiosis (2n -> 1n): homologous chromosomes segregate, programmed double strand break for cross-over

58. loss of nucleotide from degradation from break ends

o ends ligated back together -> non-homologous end-joining

o copying from a homologous duplex molecule -> homologous recombination

59. homologous recombination may result in cross-over event

o important in mitotic cells as repair mechanism => may result in loss of heterozygosity during mitotic division by cross-over between sister chromatids => one cell homozygous for normal gene and one cell homozygous for mutant gene

60. Mismatch repair (part of replication proofreading function)

o Recognize distortion in DNA, usually bound by some protein

o Complex finds a nick(differentiates new from old DNA strand) in new DNA strand, when nick found, the segment cut off and DNA synthesis in that segment is repeated

Cell cycle checkpoints – allows time for repair of DNA damage

G1 -> G1 checkpoint -> S(Intra S check point) -> G2 -> G2 checkpoint (is all DNA replicated?) -> M -> G1

If damage present, cell cycle arrests

Intra S check point – check for problem during DNA replication

Rad52 mutant -> required for repair of double strand break

Irradiate Rad52 mutant, cell does not divide and dies

Irradiate Rad9 mutant -> cell does not arrest, result microcolony but all dies

RPA(same as SSB) can sense damage in DNA

Send signals to cause response to cells

1. can recruit DNA repair pathway

2. global transcriptional response

3. DNA damage checkpoint, cell arrest to repair DNA

4. Too much damage -> apoptosis triggered

remember the syndromes and functions?????????

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LECTURE : GENE AS INFORMATION

e.g. of RNA replicating themselves Dengue, HepD

Ribosomal RNA, tRNA DNA -> RNA and stop

more universal view of information flow??

Linearity of gene

Aminoacyl tRNA synthetase – recognize shape of a.a. side chain, sense the shape of tRNA that has correct anti-codon, and charge the tRNA with a.a. => particular tRNA bound to particular amino acid

Introns – intervening sequences, not found in mature product of mRNA

Exon – anything in mature mRNA

Edges between exon and intron are well defined

300 n exon, while 3,400 n introns

Average protein coding human gene will have 8/9 exons, while 7/8 introns

Sometimes exons may be large

Can intron of one gene can be exon of another?

there's no difference in genetic code between eukaryote and prokaryote, however there maybe code usage bias between types of organisms

Globin gene/ anatomy of small gene

Promoter

Open reading frame – define something that starts with AUG and codes for x number of amino acid

Untranslated regions in mRNA -> part of first and last exons respectively

Definition of 5’ end of exon is made by where the RNA polymerase makes first copy to RNA

5’ UTR - untranslated region -> open reading frame (product begins to be made)

Splice site(SS) – edges between exons and introns

5’ SS – donor, 3’ SS - acceptor

Exons made in capital letters, introns in small letters

3’ UTR – untranslated region of last exon before its 3’ cleavage site

3’ cleavage site that defines end of last exon

Pseudogenes – unknown function

Far distant elements(locus control region, LCR)

Repeated elements in gene cluster

Long interspersed elements (LINE)

Short interspersed elements (SINE)

Most of these belong to Alu family

Very abundant throughout genome

only codon not degenerate -> AUG

what causes thalassemia? deletion of LCR that prevents synthesis of any of Beta cluster

biosynthesis of vitamin

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TEAM SESSION

watson and crick pairing vs base excision repair vs mismiatch repair vs proofreading - high fidelity

what part of DNA helix is outward? sugar-phosphate backbone

RNA used in bacterial cell wall assembly(UDP, glycan synthesis)
vitamin biosynthesis(nucleotides can be cofactors in the synthesis of vitamin)

cytosine deamination, nitric oxide vs methylation, methylation is more common common

change the transcription factor(DNA binding protein), change the of fate cell -> differentiation cascade
pluripotent - cells that are able to differentiate into many cell types
totipotent - cells that are able to differentiate into all cell types

cell type defined by switch from one pattern of regulated gene expression to another -> different transcription factors that lead to different
asymmetric cell division/distribution of content of cells -> differentiation

DNA major groove and minor groove
One groove, the major groove, is 22 Å wide and the other, the minor groove, is 12 Å wide.
The narrowness of the minor groove means that the edges of the bases are more accessible in the major groove. As a result, proteins like transcription factors that can bind to specific sequences in double-stranded DNA usually make contacts to the sides of the bases exposed in the major groove

double strand break repair

Deactivate a specific residue and see if it affects biochemical activity
- introduce wildtype and mutant type to compare function
- causation than correlation

cancer cells avoid repair pathways

ion exchange chromatography, if pH low to high, elutes negative charged particles first and then gradually toward positive charged particles; positively charged protein bound to beads of column can be eluted by increasing the concentration of sodium chloride or another salt in the buffer, sodium ion will compete with positive charged particles for the bead. Low density positive charged particle will elute first, followed by high density.