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Flashcards in Biochem Deck (59):
1

homo or heteromeric

same protein subunits, or not

2

avg weight of aa?

avg mw = 100Da

3

zwitterion

ion that carries 2 distinct ionizable groups, charge of protein depends on pH of solution. Ex: aa, has both amino and carboxyl groups that can be protonated or not

4

how is bloodstream buffered

carbonic acid and bicarbonate

5

3 sections of transmembrane/integral protein

-external domain: glycosylation and disulfide bonds
-transmembrane: highly hydrophobic alpha helix
-internal domain: reduced sulfhydryl groups, phosphorylation, no complex carb

6

ortholog vs paralog

*Ortholog=evol same across organism (bovine and human ribonuclease)
*Paralog= alteration in same protein of same organism

7

sickle cell anemia mutation

E (acidic) changes to V (neutral) in beta-globin, hydrophobic valine tries to hide, causing aggregates

8

allozymes vs isozymes

●Allozymes= forms of protein encoded by diff alleles of a gene (ie mom and dad)
●Isozymes= different proteins that all do the same thing, came from different genes (ex: CK types)

9

what is HbA1c

**real life application= adult hemoglobin is NOT N-acetylated, which renders it susceptible to N-terminal glycation. All the sugars in diabetics’ blood contain aldehyde groups, reduces unprotected valine in glucose, modified hemoglobin (HbA1c) can be measured

10

Multiple Myeloma

Ex: Multiple myeloma.
●In MM (ca of plama cells), plasma cells overproduced (primary producers of immunoglobin), immunoglobulin inc in blood detectable on gel electrophoresis.
●Treatment= velcade/Bortezomib (proteasome inhibitor) inhibits protease activity in proteasome thus blocking degradation of proteins. Myeloma cells are more sensitive to this blockage than normal cells and thus are killed more easily. (Apparently this process is still a little unclear)

11

2 types of reversible inhibition

a. Competitive: inhibitor competes with S to bind to enzyme
*Vmax doesn’t change, but amt of substrate req to reach it increases
*ex: pravastatin is competitive inhibitor, treats high cholesterol
b. Non-competitive: acts at diff site on enzyme than S. Indifferent to presence of S
*Never able to reach same Vmax as without inhibitor, but the vmax in each case (w or w/o inhibitor) can be reached with the same amt of substrate. No change in Km, does decrease Vmax

12

Irreversable inhibition

usually result of covalent modification of enzyme, cannot be overcome by inc S, can only be overcome by new protein synthesis
*Ex: PCN – covalently binds transpeptidase preventing synthesis of bacterial walls
*Ex: ASA – modifies active serine by acetylation in cyclooxygenases (COX-1 and
COX-2), preventing inflammation

13

Collagen (what is it? what tissues is it in? unusual primary sequence? unusual modification? formation of collagen?)

Collagen Example:
-long thin polypeptide made of 3 intertwined chains. Secreted protein.
-collagen is important structural protein in various tissues:
Fibril-forming
I. Skin, bone, tendon, blood vessels, cornea
II. Cartilage, intervertebral disk, vitreous body
III. Blood vessels, fetal skin
Network-forming
IV. Basement membrane
VII. Beneath stratified squamous epithelia
Fibril-associated
VIII. Cartilage
XII. Tendon, ligaments
-collagen molecules have unusual primary sequence, with lots of repeats of Gly-X-Y, X is often Pro, Y is often Hyl (hydroxyl lysine)
-collagen has an unusual modification, hydroxylation of lysines and pralines – holds collagen fibers together.

-formation of collagen: interchains assoc via disulfide bonds, wrapping begins at C-terminus¬¬¬. Tropocollagen molecules form fibrils, fibrils cross-link to form mature collagen fibers

14

Importance of vitamin C

The enzyme that catalyzes these modifications requires Vitamin C (ascorbic acid) as a cofactor. Vitamin C deficiency causes scurvy. In scurvy, assembly of collagen helices and fibers is impaired by the failure to hydroxylate prolines and lysines, and the fibers that are formed have less tensile strength than normal.

15

osteogenesis imperfecta congenital

**Osteogenesis imperfecta congenital: caused by gly bulky side chain swap within collagen fiber. Severity of disease increased if mutation is near c-term bc itll interfere with the start of wrapping.

16

quinolones

inhibit bacterial type II isomerases (diff ones in gram +/- bacteria) thus interfering with replication and transcription

17

novobiocin

aminocoumarin abx that inhibits bacterial gyrase. Typically has polycyclic ring structure.

18

daunorubicin

anti cancer = intercalate bw bases and interfers with topoisomerase activity

19

cisplatin

anti cancer = binds to bases (N7 on guanine) and crosslinks DNA, causing apoptosis

20

etoposide

chemo drug, targets mammalian topoisomerase II

21

FISH

FISH (fluorescence in situ hybridization) to identify sequences on human
metaphase chromosomes. Used by cytogeneticists to identify changes in chromosome number, translocations and indels (insertions or deletions) depending on the probes used and the magnitude of the chromosomal abnormality. Fluorescently labeled DNAs are used as hybridization probes for specific chromosomal regions in metaphase chromosome spreads.

22

rifampin/rifampicin

(abx’s)= inhibits prokaryotic RNA
polymerases by binding to it and changing its shape. (doesn’t bind eukaryotic!)

23

catabolic vs anabolic operon

-Catabolic (lac) versus biosynthetic /anabolic operon (trp – tryptophan
biosynthesis)
●Catabolic= derepressed in presence of substrate
●Biosynthetic/anabolic= repressed by product (feedback inhibition)

24

6 types of mutations

●Silent- no change in aa
●Missense- changes aa coded
●Nonsense- change aa to stop codon
●Frameshift- changes ORF, changes frames
(ex= CF)

●Also: Trinucleotide repeat expansion (ex= huntington’s, fragile X)
Splice-site mutations (ex= myotonic dystrophy)

25

selection vs screen

●selection= only phenotype of interest survives
●screen= all phenotypes survive, but phenotype of interest can be distinguished

26

cis operon vs trans operon

●cis= acting in physical proximity (ie operator acts cis to operon)
●trans=acting from a distance (ie lac repressor trans to operon)

27

Mobilization of DNA bw bacteria - transformation

uptake of naked DNA from dead donor cell, cells that can take it up are called “competent”, one strand is taken in and integrated by recombination. (Drug resistance!!)

28

Mobilization of DNA bw bacteria - conjugation

self-transmissable/mobilizable, encode structure called pilus that mediates contact and transfer. Conjugative transposons are combo of transposon and conjugation plasmid. (conjugation is significant bc natural intestinal bacteria can acquire resistance and conjugation could transfer resistance to pathogenic strains)

29

Mobilization of DNA bw bacteria - transduction

lysogenic phage acquires bits of host genome and transfers that DNA to new host

30

Lytic phage/cycle vs lysogenic phage/cycle:

Lytic =phage attaches and injects viral genome, replicated, hundreds new phages made, lysed and released
Lysogenic=integrate into target genome, can switch to lytic cycle and undergo excision to remove itself from genome. Ex: Lambda phage

31

how many bases in human genome

3.2 gigabases

32

retrotransposon, 3 types

retrotransposon= transposons that involve RNA intermediate, ie use reverse transcription to go back into the genome. EX’s:
1) Endogenous retroviruses
2) SINES (short interspersed elements): including Alu elements, 300bp full
length, Contribute to G-banding pattern of condensed x’somes, GC rich stained LIGHT
3) LINES (long interspersed elements): 5kb full length, encode structural protein, reverse
transcriptase, and endonuclease. Contribute to G-banding pattern of condensed x’somes,
AT rich fewer expressed genes and DARK

33

Modifications of N-terminal tails of histones are

1) methylation
2) acetylation [lysine]
3) phosphorylation [serine]
(Performed by methylases, demethylases, acetylases (HATs) and deacetylases (HDACs), kinases and phosphatases)

34

euchromatin vs heterochromatin

●Euchromatin: not compressed, active
●Heterochromatin: compressed, not active. 10% total DNA. Includes telomeres, centromeres, inactive X chromosome.

35

Steps of Pol II transcription

1. General transcription factors recognize and bind promoter. Ex: bind at TATA box
2. Polymerase binds
3. DNA melts at transcription site
4. Phosphorylation of carboxy-terminal domain of pol II, polymerase releases
5. Assoc of RNA processing, transcription elongation, and remodeling complexes
6. Capping 5’ end with 7-methylguanosine (Me7G)
7. RNA splicing to remove introns
8. Poly-A tail: polyadenylation of new 3’ end by adding ~200 A’s by polyA polymerase
9. Pol II termination – polymerase drops off after mRNA 3’ end processing
10. Nuclear export of mature RNA bound by export factors, cap binding factor, exon joint splicing complexes…all comprises a heterogenous ribonucleotide particle (hnRNP)

36

diseases with mutations in splicing

1) Marfan – splicing defect in fibrillin RNA
2) Muscular spinal atrophy
3) Cystic fibrosis
4) Cancer
5) Thalessemia

37

Angelman syndrome

lose one maternal copy of locus on xsome 15, or 2 copies of paternal xsome…[maternal deletion] 70%

38

Prader-Willi syndrome

deleted in paternal copy, or two copies of maternal…. [paternal deletion] 70%

39

RNA degradation
-NMD
-iron metabolism

●Nuclear degradation: incorrect RNAs targeted for exosome degradation prior to nuclear export
●Nonsense mediated decay (NMD): senses and degrades RNA’s w truncated ORFs
Medical relevance: truncated ORFS seen in ataxia telangiectasia and BRCA 1 breast
CA. about ¼ genetic mutations cld trigger NMD
●5’ and 3’ exonucleases deadenylate RNAs, susceptile to degradation
●Ex of highly regulated RNA: iron metabolism
When iron levels are low, transferrin receptor is activated by transferrin at cell surface, causes iron response elements to be stable and thus iron synthesis occurs. If there is too much iron, it will bind the iron response proteins thus causing destabilization

40

Eukaryotic non-coding RNAs (ncRNAs)

●found in nucleus= snRNA
RNA interference (RNAi) is system that helps to control which genes are active and how active they are, involved miRNA:
●found in cytoplasm= miRNA- microRNAs, short hairpin RNAs produced by Pol II that translate and degrade target RNA
1. Primary hairpin processed in nucleus by Drosha complex
2. Dicer complex then cleaves it into duplex RNA
3. One strand (called guide stand) then assimilated into RISC (RNA induced silencing complex) – that contains Argonaute proteins
●siRNA= short, synthetic RNAs designed to perform the function of the guide RNA.
Medical relevance: we can design siRNAs to target genes, has helped with therapies for AIDS, macular degeneration, etc

41

Cloning

1. Restriction enzymes cut DNA and cut vector plasmid
2. Ligated together w ligase
3. Reconstructed plasma transformed into bacteria.
4. Transformed cells selected for

42

Western blots or immunoblots

-fractionate protein mixture electrophoretically, blot to filter, probe w antibody against protein of interest, antibody/secondary antibody conjugated to radioactive, fluorescent, chromogenic reporter molecule

43

ELISA

B. ELISA (enzyme linked ImmunoSorbent Assay)
-used to detect specific protein/antibody in complex sample. Antibodies bind to antigens, antibodies contain markers. **more adaptable to clinic=not radioactive, multiple samples can be processed simultaneously

44

Southern and Northern blot

Detecting specific DNA/RNA molecules in complex mixture: “Nucleic acid blots”
-digested DNA and fractionated RNA transferred to nylon membrane. Then probes (labeled with radioactive or fluorescent tag) are used. Probes can be visualized then by exposing samples to XR film or imaging screen.
A. Southern blot: assay DNA sequences of interest, used to determine size of restriction fragment, can be used to identify insertions and deletions in genomic DNA
**can be used to identify diseases of codon repeats: Kennedy, Huntington, Fragile X, myotonic dystrophy
B. Northern blot: assay RNA

45

PCR

-amplification of specific DNA sequences in a population
-steps: melt DNA into single strand, anneal primers, extend primers using polymerization (Taq)
-med implications: prenatal testing, pathogen identification, disease diagnosis, paternity testing, forensics, and cloning
-paternity/forensics testing relies in short tandem repeats (STR) which are type of variable number tandem repeat (VTNR)
-disease testing using PCR: restriction fragment length polymorphisms (RFLPs) are linked to disease mutations. Ex: CFTR in cystic fibrosis

46

Sanger sequencing

Polymerase-based Sequencing (dideoxy or Sanger sequencing)
-polymerase-based DNA sequencing, used to sequence cloned DNA fragments
-steps: single stranded DNA to be sequenced is used as template, template made with dideoxynucleotides, split into 4 test tubes, in each tube DNA is replicated until dideoxynucleotide is taken up, these fragments can be run and gel analysis will reveal original DNA sequence

47

Microarrays (gene chips)

-contain thousands of immobilized ssDNA sequences organized onto chip. These microarrays used to analyze sample for presence of gene variations or mutations, or to determine patterns of mRNA production (gene expression analysis). Gene expression analysis – mRNA converted to cDNA and labeled w fluorescent tag, then laid over microchip, areas of brightest fluorescent correspond to highest expression of mRNA. Useful to compare two cell types, like cancer.
**depend on knowledge of sequences to be probed

48

NGS - next generation sequencing

larger scale, like entire human genome

49

2 types of starvation

●Types of starvation:
1) Marasmus starvation – total calorie undernutrition
2) Kwashiorkor – protein malnutrition, seen in children, distended abd, distorted serum aa
composition, distorted liver aa pools, albumin (edema)
3) Cachexia

50

4 toxins that interfere with making proteins

●Toxin: (will kill you in 1 week, cant go 1 week w/o making protein)
1) alpha amanitine -- inhibits RNA pol II
2) ricin – prevents binding of 60s ribosomal subunit
3) diphtheria toxin – inactivates EF2, inhibits translation
4) trichothecene mycotoxins – inhibits ribosomes, animals w hooves

51

wobble effect

Wobble effect= codon-anticodon pairing is strict for first 2 bases, last is less stringent. Significance is that there doesn’t need to be 61 codons for the aa’s, also can be used to speed up synthesis (less time to search for exact tRNA)
●loose third position
●G-U allowed
●I (or H?!) can pair with anything but G

52

shine-delgarno

Shine-Dalgarno sequence= RBS (ribosome binding site), purine-rich seq of bases 6-10 bases upstream of start codon, complementary to 3’ end of 16S rRNA (thus helping position the small ribosomal subunit)

53

polycistronic vs monocistronic

many vs one coding region, prok vs euk

54

colicin

–(antibiotic) cytotoxic ribonuclease that cleaves off 16S rRNA from A site to abolish protein synthesis

55

sulfa drugs

target methionine rxns thus blocking initiation in prokaryotic translation

56

ribozyme

acts in catalysis of peptide bond formation

57

energy per cycle of elongation?

4 high energy bonds (1 ATP and 2 GTP)

58

Streptomycin

distorts 30S subunit

59

erythromycin

blocks exit tunnel