Unit I - Cell Biology

Question 1

Intron splice site consensus sequences

Answer 1

GU marks the 5’ end of an intron

AG marks the 3’ end of an intron

Question 2

Spliceosome

Answer 2

Made of small nuclear ribonuclear proteins (snRPS) and snRNAs

U1 snRNA contains a sequence complementary to the mRNA sequence near the 5’ splice site of introns

U2 snRNA base pairs with the intron at a point corresponding to the branch point A residue, activating it for nucleophilic attack

Question 3

Purine accumulation disorders

Answer 3

Defect in hypoxanthine-guanine phosphoribosyl transferase (HGPT); leads to accumulation of purines (uric acid) in tissues

Gout
Lesch-Nyhan Syndrome

Question 4

Deamination of 5mC and C

Answer 4

Deamination of 5-methyl cytosine converts 5mC to thymine

Deamination of C yields U

Question 5

Puromycin

Answer 5

Antibiotic; inhibits bacterial translation by mimicking the 3; tRNA acceptor region and interacting with the ribosome to cause premature peptide release

Question 6

Functions of RNA Poly I, II, III

Answer 6

RNA Polymerase I - synthesizes pre-ribosomal RNA

RNA Polymerase II - synthesizes mRNA

RNA Polymerase III - synthesizes tRNA

Question 7

TATA Box / Initiator sequence

Answer 7

DNA control element located 25-30bp upstream from the transcription start site; bound by TATa-binding protein (TBP); this is the site at which general TFs bind

Question 8

TFIID

Answer 8

Contains TATA-binding protein (TBP) as well as TBP-associated factors (TAFs)

Part of RNA Polymerase II pre-initiation complex

Question 9

Rifampicin

Answer 9

Antibiotic; binds to RNA polymerase at the exit channel and sterically occludes the formation of mRNA chains > 2-3 nucleotides long

Question 10

TFIIF

Answer 10

Phosphorylates RNA Polymerase II, which activates it

Part of RNA Polymerase II pre-initiation complex

Question 11

TFIIH

Answer 11

CDK7 subunit phosphorylates Pol II CTD; has some helicase activity and functions in nucleotide excision repair (NER)

Part of RNA Polymerase II pre-initiation complex

Question 12

Nucleotide Excision Repair (NER)

Answer 12

Removes DNA lesions that distort the DNA structure (i.e. dimers) and therefore block RNA or DNA polymerase from binding

Cockayne Syndrome & Xeroderma Pigmentosum are caused by defects in NER, often TFIIH

Question 13

PolyA tail cleavage site consensus sequence

Answer 13

AAUAAA 10 to 30 nucleotides upstream from the cleavage site

Question 14

DNA Polymerase III

Answer 14

The main polymerase involved in DNA replication; Pol III “holds on” to the DNA via a sliding clamp protein and has 3-5’ exonuclease activity that enables proofreading

Question 15

DNA Polymerase I

Answer 15

Mostly involved in replacing RNA primer with DNA; has exonuclease activity in the 5-3; direction

Question 16

DNA Mismatch repair

Answer 16

MutS/MutL (in prokaryotes) or MSH/LH (in eukaryotes) recognize mismatched DNA nucleotides on the newly synthesized strand, which is unmethylated

Defects in this pathway cause hereditary non-polyposis colorectal cancer (HNPCC)/Lynch Syndrome

Question 17

Base Excision Repair (BER)

Answer 17

Removes DNA lesions that do not cause distortions in the backbone and therefore may be missed by NER; glycosylase enzymes hydrolyze the N-glycosidic bond to remove the damaged base, producing an AP site which is removed by AP-specific endonuclease

Question 18

Homologous Recombination & Non-homologous end joining (NHEJ)

Answer 18

Both repair double-stranded DNA breaks; HR repairs breaks between homologous ends; it requires the presence of a homologous chromosome (S & G2 phase)

NHEJ repairs breaks between non-homologous ends, which may lead to insertion/deletion of nucleotides at the break point

Question 19

4 categories of DNA binding domains

Answer 19

Helix-Turn-Helix (Homeodomain)
Zinc Finger
Basic Leucine Zipper Proteins (bZLP)
Basic Helix-Loop-Helix motif (bHLH)

Question 20

Histone structure

Answer 20

Histones are octameric proteins consisting of dimers of subunits H2A, H2B, H3, and H4

Histone H1 is bound to linker DNA between histones and functions to “slide” histones along DNA during modification

Question 21

Histone De/Acetylation

Answer 21

Histone Acetyltransferases (HATs) and Histone De-acetyltransferases (HDATs) are classes of reversible chromatin modifiers; acetylation of histone N-termini Lysine residues neutralizes the positive charge and “frees” the histone tails from electrostatic reactions with the DNA phosphate backbone

Question 22

Stop Codons

Answer 22

UAA
UAG
UGA

Question 23

Shine-Delgarno Sequence

Answer 23

Prokaryotic ribosomal binding site; essentially corresponds with initiator AUG codon

Question 24

Initiation Factor 4E (IF4E)

Answer 24

Binds the 7-methyl guanosine cap on the 5’ end of the mRNA, facilitating scanning of the ribosome down the primary transcript to the start AUG codon

Question 25

Elongation Factors (Prok and Euk)

Answer 25

Ef-Tu (in bacteria) & Ef1A (in eukaryotes); deliver charged aa-tRNA complexes to the A site of the ribosome, burning GTP when it dissociates

Question 26

Initiation of Translation - Bacteria

Answer 26

IF1 and IF3 bind the small subunit, which binds the mRNA at the AUG start codon via base pairing with the Shine Delgarno sequence IF2 (bound to GTP) delivers initiation Met-tRNA to the P site to pair with the AUG codon GTP hydrolysis on IF2 releases all IFs and signals binding of the large subunit

Question 27

DNA Polymerase delta

Answer 27

Performs DNA synthesis on the lagging strand in eukaryotes; contains 3-5' exonuclease activity

Question 28

DNA Polymerase alpha

Answer 28

Functions in the synthesis of the first ~20 DNA nucleotides immediately following the RNA primers; also synthesizes the first basepairs of each Okazaki fragment DNA polymerase alpha has primase activity

Question 29

DNA Polymerase epsilon

Answer 29

Performs DNA synthesis on the leading strand in eukaryotes; contains 3-5' exonuclease activity

Question 30

Release Factor (RF)

Answer 30

Enters the A site (bound to GTP) in conjunction with a stop codon; hydrolysis of GTP induces release of the ribosomal subunits

Question 31

Sources & Types of High Energy Bonds

Answer 31

``` Thioester bonds (i.e. Acetyl coA) Phosphoanydride bonds (i.e. ATP) Phosphoenolpyruvate (PEP) ```

Question 32

Relationship between free energy, enthalpy, and entropy of a system

Answer 32

dG = dH - TdS

Question 33

Reduction Potential (E)

Answer 33

A measure of the willingness of a molecule to become reduced; the more positive the value of E, the more likely a molecule is to accept electrons (become reduced); the more negative the value of E, the more likely a molecule is to donate electrons (become oxidized) Energy is released as electrons pass from compounds with low E to high E values

Question 34

Relationship between free energy and redox potential

Answer 34

dG = -nFdE

Question 35

Enzyme cofactor vs. coenzyme

Answer 35

Cofactors are small molecules (often metal ions) that function with an enzyme to catalyze a reaction; cofactors are not used up in the reaction Coenzymes provide chemical groups for a catalyzed reaction; they are used up by the reaction Prosthetic groups are cofactors/coenzymes that are tightly bound to the enzyme

Question 36

Km

Answer 36

Km is the substrate concentration at which Vo = 1/2(Vmax) Km typically approximates intracellular concentrations of [S]

Question 37

Kcat

Answer 37

Kcat describes the number of substrate molecules turned into product per enzyme molecule, per second, at saturating substrate concentrations The larger the Kcat value, the faster the enzyme works to turnover

Question 38

Kcat/Km

Answer 38

A measure comparing enzyme efficiency; the larger the value of Kcat/Km, the more efficient the enzyme Enzymes that are "catalytically perfect" have a Kcat value approaching 10^8; the enzyme is so efficient that rate of diffusion is the only limiting factor

Question 39

Inhibitors - 3 classifications

Answer 39

Competitive - Bind to the enzyme active site, blocking binding of the natural substrate Uncompetitive - Bind to the ES complex at a site other than the active site Mixed - May bind to either E or ES at a site other than the active site

Question 40

Alpha Helix

Answer 40

H-bonds form between the carbonyl oxygen of the nth AA and the amide nitrogen of the n + 4th AA within the same polypeptide chain, forming a right-handed screw Ala and Leu are commonly found; Pro and Gly do not occur

Question 41

Beta Sheets

Answer 41

Secondary structure motif formed by hydrogen bonds between two polypeptide chains running parallel or anti-parallel to each other Ex: Immunoglobulin, antibodies

Question 42

Kd

Answer 42

Dissociation constant; Kd is the ligand concentration at which 50% of the protein is ligand-bound Smaller Kd value implies a stronger binding interaction

Question 43

Myoglobin

Answer 43

Stores oxygen; oxygen binds Fe2+ in the heme group, which is buried within the myoglobin protein; Kd is very low, so myoglobin stores oxygen but does not transport

Question 44

Hemoglobin

Answer 44

Hb has 4 O2 binding sites that interact with allostery; O2 binding triggers a T to R conformational state change in which Hb has higher affinity for O2 Hb binds O2 better under local conditions of higher pH (i.e. in the lungs) and releases O2 under conditions of lower pH (i.e. in metabolically active tissues)

Question 45

2 Classes of Chaperone Proteins

Answer 45

1. HSP70 - binds to the hydrophobic regions of unfolded proteins, preventing aggregation and facilitating re-folding 2. GroEl/GroES (prok) / HSP60 (euks) - forms a capped, barrel-like structure that isolates mis-folded proteins and hydrolyzes ATP to aid in re-folding

Question 46

Alzheimers - 3 primary genetic pathways

Answer 46

1. Missense mutation in APP gene causes increased production of all B-amyloid proteins, including B-42 via cleavage by B-secretase and y-secretase 2. ApoE4 genetic variant leads to decreased AB 42 clearance 3. Mutation in presenelin genes, leading to increased production of AB42 (more common in familial cases of Alzheimers)

Question 47

Stages of the Cell Cycle

Answer 47

G1 - Cell growth; building and loading of Pre-RC onto origin S - Activation of Pre-Rc, DNA Synthesis G2 - Protein synthesis and cell growth M - Chromosome Segregation

Question 48

Rad 17, ATM/ATR, p53, p21, Rb cycle

Answer 48

Rad17 senses & binds to damaged DNA, recruiting ATM/ATR ATM/ATR proteins kinases phosphorylate p53 Phosphorylated p53 is a TF that promotes synthesis of p21, a CDK inhibitor p21 inhibits CDK, which fails to phosphorylate Rb Unphosphorylated Rb binds to and inhibits transcription factor E2F, which is necessary for transcription of a group of genes involved in DNA synthesis In response to DNA damage the cell is held in G1 phase and cannot progress to S phase

Question 49

Pre-RC Cycle

Answer 49

Pre-RC is built and loaded onto replication origins in G1; Pre-RC are activated during S phase This ensures that cells may not replicate and divide during the same phase of the cell cycle

Question 50

VDJ Recombination

Answer 50

An endogenous source of double stranded DNA breaks responsible for generation of diversity in immunoglobulin DNA via non-homologous end joining; mutations in NHEJ genes lead to severe combined immunodeficiency (SCID) because functional B-cells and T-cells are not produced

Question 51

Crohn's Disease

Answer 51

Most often affects the Ileum & Colon, although may affect the entire GI tract Characterized by: discontinuous lesions, transmural inflammation, perianal disease, strictures & fistulas

Question 52

Ulcerative Colitis

Answer 52

Most often affects the colon & rectum Characterized by: continuous lesions, mucosal inflammation, hematochezia

Question 53

Extra-GI manifestations of Crohn's / Ulcerative Colitis

Answer 53

Infections of the eyes - Iritis, episcleritis Infections of the lungs & heart - pleuritis, myocarditis Pancreatitis Ankylosing Spondylitis (chronic inflammatory arthritis) Sacroileitis Erythema Nodosum - inflammation of subcutaneous fat (usually on shins) Pyoderma gangrenosum (chronic ulcers on the legs)

Question 54

Swi/Snf

Answer 54

Chromatin remodeling complex that uses DNA to break histone/DNA contacts and slide the histones down the DNA, "loosening" the chromatin structure

Question 55

Tetracycline, Streptomycin, Erythromycin

Answer 55

Antibiotic drugs that bind to the small subunit of the prokaryotic ribosome, preventing entry of aa-tRNAs into the A site and thus inhibiting bacterial protein synthesis

Question 56

Interferon

Answer 56

Interferon is released by cells in response to viral infection; in neighboring cells, it stimulates the activity of kinases which phosphosrylates eIF2; when phosphorylated, eIF2 is bound by eIF2-BP and initiation of translation is stalled

Question 57

eIF2

Answer 57

A eukaryotic initiation factor required for translation; eIF2 mediates the binding of the initiator t-Met complex to the ribosomal P site; after formation of the proper codon-anticodon pairing, eIF2 hydrolyzes GTP and releases from the ribosome

Question 58

Ciclosporin

Answer 58

An immunosuppressant drug; ciclosporin binds to cyclophilin, inhibiting activation of calcineurin; normally, calcineurin functions to dephosphorylate the transcription factor NFAT, allowing it to move to the nucleus and activate transcription of genes coding for immune cell proliferation; ciclosporin therefore prevents translocation of the TF NFAT

Question 59

Rubenstein-Taybi Syndrome

Answer 59

Results from mutations in one copy of the CREB-BP gene; CREB-BP is a transcriptional co-activator for many TFs and is a HAT; presents as cognitive disability & craniofacial dysmorphism

Question 60

a-aminitin toxin

Answer 60

Death cap mushroom toxin; blocks the translocation of RNA polymerase II by interacting with its bridge helix

Question 61

Tamoxifen

Answer 61

Tamoxifen antagonizes estrogen by binding to the estrogen receptor (ER) and preventing recruitment of HAT co-factors (activators)

Question 62

NFKB Pathway

Answer 62

NFKB protein is usually held in the cytoplasm by binding to its inhibitor (IKB); phosphorylation of IKB targets it for degradation, releasing NFKB to move into the nucleus where it turns on its target genes, including genes involved in inflammation Aspirin inhibits the phosphorylation of IKB

Question 63

Rapamycin

Answer 63

An immunosuppressant drug; Inhibits mTOR (mammalian target of Rapamycin), which is a kinase that phosphorylates 4E-BP; inhibition of mTOR leads to dephosphorylation of 4E-BP, which is the state in which it binds eIF4E, silencing translation