Exam 1 Biochemistry Thread

Question 1

Gibbs free energy (G)

Answer 1

The potential energy of a chemical reaction or system with the capacity to do work.

Question 2

High Energy Bond

Answer 2

Any bond that releases > -7 kcal/mol upon dissociation.

Question 3

Gibbs Free Energy Equation

Answer 3

The change in free energy of a system (∆G) at constant pressure and temperature.

∆G = ∆H - T∆S

∆G ⇒ change in free energy

∆H ⇒ change in enthalpy

∆S ⇒ change in entropy

T ⇒ absolute temperature in Kelvin

Question 4

Reaction Spontaneity

Answer 4

∆G > 0 ⇒ Reaction proceeds in the reverse direction (endergonic)

∆G = 0 ⇒ equilibrium

∆G < 0 ⇒ Reaction proceeds in the forward direction (exergonic)

Question 5

Equilibrium Constant

(K or K_eq)

Answer 5

Defined as the ratio of the concentration of products to concentration of reactants.

A + B ↔︎ C + D

Question 6

Free Energy Change

Answer 6

The free energy change for a reaction (∆G) is given by:

Question 7

Standard Free Energy

At Equilibrium

Answer 7

∆G = 0.

Concentrations of reactants and products are at equilibrium values.

Standard free energey change is related to the equilirium constant (K_eq).

Question 8

Reaction Kinetics

Answer 8

Rate of ractions determined by the rate constants (k) and concentrations of reactants.

Equilibrium occurs when the rate of the forward reaction equals the rate of the backwards reaction.

Question 9

∆G Trends

Answer 9

As we approach equilibrium:

K_eq gets bigger because [products] > [reactants]

∆G gets smaller

More negative K ⇒ more positive ∆G.

Question 10

D & L

Isomers

Answer 10

D & L isomers are enantiomers.

D if OH on the farthest chiral C is on the right

L if OH on the farthest chiral C is on the left

D form in the body.

Question 11

α and β

Isomers

Answer 11

α and β isomers are the result of ring formation ⇒ anomers

Fischer projections:

alpha ⇒ OH on the same side as oxygen ring

beta ⇒ opposite side

Hayworth projections:

alpha = trans

beta = cis

Question 12

Fisher to Hayworth

Projections

Answer 12

right side ⇒ up

left side ⇒ down

Question 13

Anomeric C

Answer 13

The anomeric C is linked to two oxygens.

C#1 → anomeric C in aldoses

C#2 → anomeric C in ketoses

Question 14

Epimers

Answer 14

Isomers that differ in the position of OH at only one carbon.

Considered diasteriomers.

Question 15

Galactose

Answer 15

C4 epimer of glucose.

Question 16

Mannose

Answer 16

C2 epimer of glucose

Question 17

Reducing Sugar

Answer 17

Anomeric C has available OH.

All monosaccharides are reducing sugars.

Ketose must tautomerize to an aldose first.

Question 18

Keto-Enol Tautomerization

Answer 18

Sugars freely interconvert between the keto and aldo forms in solution.

Catalyzed by base.

Question 19

Mutarotation

Answer 19

Cyclic sugars undergo epimerization between the alpha and beta anomeric forms in solution.

Forms a racemic mixture ⇒ racemization.

Shows mutarotation of plane polarized light.

Question 20

Glucose Test

Answer 20

Urine test for glucose:

Glucose oxidase used to oxidize glucose to gluco-lactone and H₂O₂.

Peroxidase used to visualize the H₂O₂.

Question 21

Clinitest

Answer 21

Uses Benedict’s reagent (copper reduction) to test for the presence of reducing sugars in urine.

Question 22

Monosaccharide Reactions

Oxidation of terminal OH group

Answer 22

Question 23

Monosaccharide Reactions:

Reduction of Carbonyl C

Answer 23

Yields new OH and creates a polyol.

Question 24

Monosaccharide Reactions:

Reduction of OH

Answer 24

Reduction of OH on C#2 yields a deoxy sugar

Question 25

Monosaccharide Reactions: Replacement Reactions

Answer 25

Replacement of OH (usually at C#2) yields an amino sugar. NH₂ can then be acetylated.

Question 26

Activated Monosaccharides

Answer 26

Monosaccharides activated as first step in metabolism.

Question 27

Glycosides

Answer 27

Monosaccharides can be linked together via **glycosidic bonds** to form **glycosides**. Glycosidic bonds form by **glycosyltransferases** via **condensation** of OH on anomeric C with OH of another sugar. Glycosidic bonds cleaved by **glycosidases** and **glycosylases**.

Question 28

Glycosidic Bonds

Answer 28

α linkages → cis β linkages → trans

Question 29

Lactose

Answer 29

galactosyl-β (1→4)-glucose

Question 30

Sucrose

Answer 30

glucosyl - α (1→2) - fructose

Question 31

Maltose

Answer 31

glucosyl - α (1→4) - glucose

Question 32

Glycogen

Answer 32

Storage form of glucose in animals. Branched chain polymer of α-D-glucose. α 1→4 chains α 1→6 branches

Question 33

Starch

Answer 33

Storage form of glucose in plants. Contains: Unbranched polymer of α-D-glucose via α 1→4 linkages ⇒ **amylose** & Branched polymer of α-D-glucose α 1→6 linkages ⇒ **amylopectin**

Question 34

Cellulose

Answer 34

Structural component of plant cell walls. Unbranched polymer of β-D-glucose via β 1→4 linkages.

Question 35

Glycoproteins

Answer 35

Proteins with short, often branched chains of oligosaccharides attached.

Question 36

Proteoglycans

Answer 36

Large complexes of negatively charged **heteropolysaccharides** associated with a small amount of protein (**core protein**). Important components of the extracellular matrix. Large negative charge favors extended conformation and extensive hydration ⇒

Question 37

GAGs

Answer 37

Proteoglycan containing **glycoaminoglycan** (GAG) a.k.a mucopolysaccharides Long, unbranched, negatively charged. Contains a repeating unit **[acidic sugar-amino sugar]_n** Usually [glucuronate glucosamine]. Glucosamine is frequently **acetylated** and often **sulfated** to increase negative charge.

Question 38

Chondroitin Sulfates

Answer 38

The most abundant proteoglycan GAGs in humans. Contains the repeating unit: [D-glucuronate - N-acetyl-D-galactosamine-6(4)-sulfate]_n

Question 39

Bonds in DNA

Answer 39

Sugar and phosphoryl group linked via an **ester bond.** Nucleotide monophosphate (NMP) monomers linked by **3'-5' phosphodiester bond.** Nitrogenous base linked to sugar via **N-glycosidic bond.**

Question 40

Base Pairing

Answer 40

Nitrogenous bases of DNA linked via hydrogen bonds. Adenine - Thymine stabilized by 2 H-bonds. Guanine - cytosine stabilized by 3 H-bonds.

Question 41

DNA Secondary Structure

Answer 41

Twisted right-handed helix * Base stacking decreases contact between water and hydrophobic face ⇒ **hydrophobic effect** * **Van der Waals forces** further stabilize the base * Forms major and minor grooves

Question 42

DNA Denaturation

Answer 42

* Alkali treatment (pH \> 11.3) * Cause H-bonds to break * Does not destroy phosphodiester bonds * Heat * Midpoint of heat denaturation (T_m) correlated with GC/AT ratio

Question 43

DNA Renaturation

Answer 43

Denatured DNA can realign and base-pair. Denatured DNA can undergo **hybridization** and anneal with complementary mRNA strands.

Question 44

Forms of DNA

Answer 44

* B - DNA * normal DNA * R-handed helix * predominants * A - DNA * DNA / RNA hybrid * Z - DNA * L-handed helix * via stretches of alternating purine and pyrimidine nucleotides A-DNA and Z-DNA are rare and thought to be involved in regulation of transcription.

Question 45

mtDNA

Answer 45

Double stranded Circular Codes for 13 proteins. Found in multiple copies in the mitochondrial matrix.

Question 46

Nucleohistone

Answer 46

Consists of DNA and histones. **[H2A, H2B, H3, and H4]₂** forms octomer **nucleosome core** **H1** binds to linker DNA acts as **linkages** Forms beads on a string structure. Nucleosomes wind into coils ⇒ **nucleofilaments** / solenoid structures Solenoid structures further compact forming chromosomes.

Question 47

mRNA Structure

Answer 47

* Has considerable secondary and tertiary structure due to loop-like structures within RNA single strand * When 2 regions of ssRNA complementary, can base pair to form stem-loop or hairpin structure.

Question 48

tRNA Structure

Answer 48

* High percentage of modified bases * 2º structure ⇒ cloverleaf * Base pairing occurs in stem regions resulting in ds regions * 3º structure ⇒ inverted "L" * D-loop ⇒ contains dihydrouridine (D) * **Anticodon loop** ⇒ base-pairs with codon on mRNA * TψC loop ⇒ contains ribothymidine (T) and pseudouridine (ψ) * Variable loop ⇒ varies in size * **3' CCA sequence** ⇒ attachment site for amino acid

Question 49

Ribozyme

Answer 49

RNA with catalytic activity. Ex. rRNA ⇒ forms peptide bond linking AA in proteins

Question 50

Peptide Bond

Answer 50

* Amide bond links AA in a protein ⇒ peptide bond * Acts as a double bond due to resonance * Planar

Question 51

Protein Bond Angles

Answer 51

* omega (ω) angle ⇒ peptide bond ⇒ 0º or 180º * phi (φ) angle ⇒ nitrogen and alpha carbon * psi (ψ) angle ⇒ carbonyl carbon and alpha carbon If phi and psi angles for the whole protein is known, the 3-D structure of the protein is also known.

Question 52

Beta Strand

Answer 52

2º structure of protein Elongated straight chain stabilized by **hydrogen bonding across the backbone** (i.e. perpendicular). H-bonds can be **intra-chain or inter-chain**. Same orientation ⇒ **parallel** Opposite orientations ⇒ **anti-parallel** Can zig-zag to form **β pleated sheets**.

Question 53

α-Helix

Answer 53

2º structure of protein Stabilized by **intra-chain** hydrogen bonds **parallel** to central axis. Cannot incoorporate **proline** residues.

Question 54

β-Turn

Answer 54

2º structure of protein Loop in the protein. Stabilized by **backbone** hydrogen bond that causes chain to **change direction**. Typically involves **four amino acids** including a **pro** and **gly**.

Question 55

DNA-Binding Motifs

Answer 55

* Helix-loop-helix (dimer) * Tandem array of Zinc Fingers * Leucine zipper (dimer) * Homeodomain (contains helix-turn-helix) * From homeobox complex with DNA

Question 56

Protein Folding

Answer 56

* Starts immediately after translation * Largely dictated by the amino acid sequence * Forms molten globule structure * Added by chaperones * Ex. GroEl

Question 57

Protein Quarternary Structure

Answer 57

Arrangement of multiple folded protein subunits to form a multi subunit complex. Usually bind via non-covanlent interactions: hydrophobic interactions hydrogen bonding electrostatic interactions

Question 58

Prosthetic Groups

Answer 58

Non-amino acid groups required by a protein to be functional.

Question 59

Protein Isoforms

Answer 59

Protein with the same function as another protein but which is encoded by the same or a differnt gene and may have small differences in sequence.

Question 60

Amino Acid Basic Structure

Answer 60

* At physiological pH 7.4, carboxyl group dissociated and amino group protonated * α-carbon is **chiral** except for glycine * **L-form** found in mammals * R-group determines solubility

Question 61

Non-Polar Amino Acids

Answer 61

**GLAM VIP** **F**or **W**omen

Question 62

Polar Uncharged Amino Acids

Answer 62

**Q**ueens **N**ever **T**ake **Y**our **S**illy **C**rown

Question 63

Polar (+) Charge Amino Acids

Answer 63

HRK

Question 64

Polar ( - ) Charge Amino Acids

Answer 64

Question 65

Cystine

Answer 65

2 cysteines form a covalent disulfide bond ⇒ cystine

Question 66

Histidine

Answer 66

Histidine's heterocyclic imidazole side chain weakly basic and largely uncharged at physiological pH. When incorporated into a protein, can be neutral or positively charged depending on environment. R group has a pI ~ 6.5-7.4 in proteins meaning it can serve as a **buffer** at physiological pH.

Question 67

Essential Amino Acids

Answer 67

HV MILK FTW Histidine Valine Methionine Isoleucine Leucine Lysine Phenylalanine Threonine Tryptophan

Question 68

Isoelectric Point (pI)

Answer 68

The pH at which the net charge on a molecule is zero. The average of the pK's for amino acids with 2 ionizable groups: pK₁ for the α-carboxyl group ~ 2-3 pK₂ for the α-amino group ~ 9-10 The average of the two pK's on either side of the isoelectric form for those with 3 ionizable groups.

Question 69

Plasma Protein Electrophoresis

Answer 69

Carried out at pH above pI values for most plasma proteins. Proteins will carry a **negative charge** and move towards the **positive electrode** at a rate dependent on their net charge.

Question 70

Apoenzyme

Answer 70

The inactive enzyme without its coenzyme.

Question 71

Holoenzyme

Answer 71

The active form of an enzyme with a coenzyme component.

Question 72

Co-factor

Answer 72

A **metal ion** coenzyme.

Question 73

Coenzyme

Answer 73

A **small organic molecule** coenzyme.

Question 74

Cosubstrates

Answer 74

Coenzymes that are **loosely** associated with the enzyme and **leave in a changed state.**

Question 75

Prosthetic Group

Answer 75

Coenzyme that is **permanently attached** and **is not altered** during the reaction.

Question 76

Classification of Enzymes

Answer 76

Question 77

Michaelis Menten Equation

Answer 77

Describes the kinetics of enzyme-catalyzed reactions. **V_max** ⇒ the maximal rate of the reaction when all of the enzyme is occupied by substrate. **K_m** is a reflection of the enzyme's affinity for substrate. (Not when K_cat is included) When **[S] = K_m ⇒ v = 1/2 V_max**

Question 78

Turnover Rate | (K_cat)

Answer 78

**The maximum number of substrate molecules converted to produce per enzyme molecule per second.** Measures the efficiency of the enzyme. Theoretical upper limit of 10⁹ M^-1sec^-1 ⇒ catalytic perfection Reaction becomes diffusion limited.

Question 79

Lineweaver-Burke

Answer 79

Linearized version of the Michaelis-Menten Equation

Question 80

Irreversible Inhibition

Answer 80

The destruction or modification of one or more functional groups of the enzyme resulting in a permanent reduction in enzyme activity. Decreases V_max.

Question 81

Reversible Inhibition

Answer 81

Takes place only in the presence of a specific inhibitor. Competitive inhibition vs Noncompetitive inhibition

Question 82

Competitive inhibition

Answer 82

Inhibitor binds to the active site Can be reversed by increasing [substrate] **No change in V_max** **Apparent increase in K_m**

Question 83

Noncompetitive Inhibition

Answer 83

Inhibitor binds somewhere other than the active site. ## Footnote **No change in K_m.** **Apparent decrease in V_max.**

Question 84

Allosteric Regulation

Answer 84

Effector or ligand binds to the regulatory site. Enzymes often have more than one subunit. Shows a **sigmoidal** *v* vs [S] curve. Increase activity ⇒ **positive allosteric effector** Decrease activity ⇒ **negative allosteric effector** Can alter K_m, V_max, or both. **Homotrophic** **effector** ⇒ substate for that enzyme **Heterotrophic** **effector** ⇒ seperate molecule

Question 85

Covalent Modification of Enzymes

Answer 85

Regulation of enzymes via reversible covalent modification. Most often phosphorylation/dephos at serine, threonine, or tyrosine residues.

Question 86

Prokaryotic DNA Replication

Answer 86

1. **Helicase** seperates DNA strands at the AT rich '_origin_' of replication. * **Topoisomerase** relieves supercoils * **Single strand binding proteins** prevent reannealing 2. **DNA primase** lays down an RNA primer 3. **DNA polymerase III** replicates DNA continuously in 5'→3' direction on the _leading strand_ and in Okazaki fragments in the 3'→5' direction on the _lagging strand_. * **Sliding clamp complex** holds DNA pol on the DNA ⇒ **processivity** 4. **DNA polymerase I** degrades RNA primer and fills in with DNA. 5. Fragments joined by **DNA ligase**. \*DNA pol II with repair function.

Question 87

Eukaryotic DNA Replication

Answer 87

1. **Origin Recognition Complex (ORC)** binds to the origin of replication. 2. ORC recruits **cell division cycle 6 (CDC6)** to coordinate replication with mitosis. 3. **Minichromosome maintenance complex (MCM)** acts as *helicase* to seperate DNA strands. (ATP dependent) * **Topoisomerase** undoes supercoils formed * **Replication Protein A (RPA)** [*SSB*] prevents reannealing 4. **DNA polymerase α** lays down RNA primer. 5. **DNA polymerase ε** (epsilon) elongates _leading strand_. * *DNA polymerase δ** (delta) elongates _lagging strand_. * Both have 3'→5' exonuclease activity for proofreading. * **Replication Factor C (RFC)** [*clamp loader*] loads **Proliferating Cell Nuclear Antigen (PCNA)** [*clamp*] which holds Pol on the DNA ⇒ _processivity_ 6. **RNase H1** or **Fen1** degrades RNA primer on lagging strand. * Fen1 has 3'→5' exonuclease activity for proofreading. 7. **DNA polymerase δ** fills in the gap with DNA. 8. **DNA ligase I** joins Okasaki fragments.

Question 88

Telomeres

Answer 88

Non-coding **hexanucleotide repeats** (AGGGTT) at the ends of linear chromosomes that maintain the structural integrity of eukaryotic chromosomes. **Lagging strand end problem** arises with removal of RNA primer during synthesis. Shorter and shorter daughter DNA molecules with **incomplete 5' ends** result with each round of replication.

Question 89

Telomerase

Answer 89

**Ribonucleoprotein** present in constantly replicating cells. Addresses lagging strand end problem. "Template-bearing reverse transcriptase" * Telomerase has **RNA molecule** that acts as a template for **telomere elongation**. * Telomerase has **polymerase activity** that places new telomere repeat at the 3' end of leading strand. * **DNA pol α** inserts RNA primer on the 5' end of lagging strand and elongates in the 5'→3' direction. * RNA primer removed after.

Question 90

Prokaryotic Mismatch Repair (MMR)

Answer 90

**Corrects single base mismatches or small insertions/deletions.** * **MutS** dimer follows behind DNA Pol looking for _distortions_ in helix * **Mut S** binds to mutated DNA and _recruits MutH and MutL_ * **MutH** r_ecognizes methylated parent_ strand * **MutL** _activates_ the complex to _form a loop_ * **MutH** _nicks daughter strand_ near methylated site and _recruits a helicase_ * Complex _slides past site_ of mismatch followed by **exonuclease**, leaving gap extending beyond mismatch. * _Gap filled_ by **DNA Pol III** * _Sealed_ by **DNA ligase**

Question 91

Eukaryotic Mismatch Repair (MMR)

Answer 91

Corrects single base mismatches or small insertions/deletions. * **MutS** and **MutSβ** recognize mismatch, bind, and recruit complex. * **MutLα** binds and activates **exonuclease** activity removing mismatched base * Synthesis of repaired strand requires **RFC** (clamp loader), **PCNA** (sliding clamp), **Pol-δ**, and **DNA ligase**.

Question 92

Microsatellite Instability

Answer 92

Microsatellites are tandem repeats of 2-5 base pairs x thousands. Microsatellite instability is **genetic hypermutability due to defect in MMR.** Can cause **Hereditary Non-Polyposis Colorectal Cancer** aka **Lynch syndrome**.

Question 93

Nucleotide Excision Repair | (NER)

Answer 93

**Corrects damaged DNA containing bulky adducts.** Ex. Thymine dimers from UV damage or chemical carcinogens * Region of damaged strand _excised_ by **excision endonuclease** (aka exinuclease) * Hydrolyzes phosphodiester bonds on both sides of the lesion * **DNA Pol** copies undamaged strand with help from **RFC** and **PCNA** * **DNA ligase** seals gap Xeroderma pigmentosum and basal cell carcinomas result from defects.

Question 94

Base Excision Repair | (BER)

Answer 94

Used to repair damaged single bases (non-bulky damage) caused by oxidation, alkylation, hydrolysis, or deamination. * **Glycosylase** _cuts N-glycosidic bond_ of damaged base and removes base * Leaves either apyrimidinic or apurinic site * **Endonuclease** _removes sugar_ * **Lyase** _removes phosphate_ * **DNA Pol** and **ligase** _finishes repair_

Question 95

Homologous Recombination | (HR)

Answer 95

**Uses homologous dsDNA as template ⇒ error-free** * Broken dsNDA processed by **exonucleases** to produce DNA duplex with a **protruding 3'-ssDNA tail** * **RAD51**, _homologous pairing protein_, loaded onto ssDNA * RAD51 searches for an _intact homologous DNA template_ * Template used for complementary DNA synthesis

Question 96

Non-homologous ends joining | (NHEJ)

Answer 96

Repairs DS breaks in template-independent manner ⇒ error prone. Some DNA lost. * **KU proteins** and **DNA-PKcs** (DNA-dependent protein kinases) recognize and link broken DNA ends * Recurits **nucleases** to make ends better substrates for ligation * **Ligases** connect ends

Question 97

DNA ⇒ RNA Template

Answer 97

RNA produced is complimentary to the **template strand**. The non-template strand is identical (except for U's) to the RNA produced ⇒ called the **coding strand**.

Question 98

Ribosomal RNA | (rRNA)

Answer 98

Major building blocks of ribosomes. Prokaryotes: 5s, 16s, 23s Eukaryotes: 5s, 5.8s, 18s, 23s

Question 99

Small Non-coding RNAs | (ncRNA)

Answer 99

Involved in regulation and are not translated into proteins. Three main types: 1. Small nuclear RNAs (snRNAs) 2. Small nucleolar RNAs (snoRNAs) 3. MicroRNAs (miRNA)

Question 100

Small nuclear RNAs | (snRNAs)

Answer 100

Used in the spliceosome.

Question 101

Small nucleolar RNAs | (snoRNAs)

Answer 101

Guide chemical modifications of rRNA, tRNA, and snRNA.

Question 102

MicroRNAs | (miRNA)

Answer 102

Function in RNA sliencing and post-transcription regulation of gene expression.

Question 103

Prokaryotic Transcription

Answer 103

* **_Initiation_** * Holoenzyme of RNA polymerase (**core + sigma factor**) recognizes and binds to the -**35** consensus sequence and inserts into DNA at the **-10 TATA box** of the promotor ⇒ **closed complex** * **Hydrolysis of ATP** activates **helicase** activity of RNA pol seperating DNA ⇒ **open complex** * RNA pol makes several abortive attempts, **sigma factor dissociates**, and **elongation** **begins**. * **_Elongation_** * Ribonucleotides added to 3' end of RNA transcript * RNA pol does not have exonuclease activity but will halt at mismatched base-pairs and remove it * **_Termination_**: two methods * **Intrinsic termination sites** * Palindromic GC-rich region followed by oligo U's on nascent mRNA * Forms **stem-loop** that interacts with RNA pol causing it to pause and dissociate * Oligo U's after stem-loop cause nascent RNA transcript to dissociate from DNA * **Rho-dependent** * Nascent mRNA has 5' Rho binding site * **Rho factor** *[RNA-dependent ATPase with helicase activity]* binds to mRNA * Rho moves up mRNA and strips it from RNA Pol and DNA

Question 104

Prokaryotic mRNA

Answer 104

* 5' UTR or leader sequence (Shine-Delgarno Sequence) ⇒ recognizes ribosome for translation * AUG start codon codes for N-formyl-Met * 3' UTR or trailing sequence * Polycistronic

Question 105

Chromatin Modification

Answer 105

* **Histone acetyltransferases (HATs)** * **Transfer acetyl group** to 1º amine of lysine side chain on histone ⇒ **neutralizes** charge-charge interaction * Works with chromatin remodelers to form **euchromatin** * **Histone deacetyltransferases (HDACs)** * **Removes acetyl group** from histones * Works with chromatin remoders to change euchromatin to **heterochromatin**

Question 106

Nucleosome and REmodeling and Deacetylase Complex (NuRD)

Answer 106

ATP-dependent chromatin recomdeling complex. Has histone binding proteins and deacetylase activity. Important role in regulation of gene transcription, genome integrity, and cell cycle regulation.

Question 107

RNA Polymerase I

Answer 107

* Found in the **nucleolus** * **Synthesizes rRNAs except 5S rRNA** * Recognized by general transcription factors (GTFIs) * Regulation linked to cell growth and protein translation

Question 108

RNA Polymerase II

Answer 108

* Synthesizes mRNA * Synthesizes most small noncoding RNAs * Has a wide variety of regulatory elements * Most controlled Pol

Question 109

RNA Polymerase III

Answer 109

* Makes **tRNA** * Makes **5S rRNA** * Makes **some snRNA and snoRNA** * Most are 'housekeeping' genes needed all the time * Regulation tied to cell growth and cell cycle * Requires fewer regulatory proteins compared to RNA Pol II * **Requires no control sequences upstream of the gene** * Relies on **internal control sequences** within the transcribed gene

Question 110

Core Promoter

Answer 110

**The minimal DNA sequence required to direct the initiation of transcription.** **Region where RNA polymerase and general transcription factors interact.** No consensus sequence in eukaryotes. Ex. TATA box or Hogness Box

Question 111

General Transcription Factors | (GTFs)

Answer 111

aka basal transcription factors **The most basic set of proteins needed to activate gene transcription.** **Binds to the template DNA.** Help to **localize** **RNA polymerase** and regulation initiation of elongation.

Question 112

Proximal Control Elements

Answer 112

DNA sequences within 250 base pairs of the start site that alter the rate of transcription initiation by interacting with regulators. Are typically position dependent. Usually upstream. Ex: CAAT box ⇔ CAAT box transcription factor (CTF) GC box ⇔ Sp1

Question 113

Distal Control Elements

Answer 113

DNA sequences which are farther away from the start site that alter transcription. Position and orientation independent. Can be enhancers or silencers.

Question 114

Specific Transcription Factors | (STFs)

Answer 114

Interact with proximal and distal control elements to modulate the level of transcription. **DNA binding domain (BD)** ⇒ recognizes the control elements **Transactivation domain** ⇒ binds chromatin remodeling factor, co-activators, or co-repressors, and other factors.

Question 115

Mediator Proteins

Answer 115

Forms a complex that acts as a bridge betwee the regulators and core promoter.

Question 116

RNA Polymerase II Pre-initiation Complex

Answer 116

* **TATA Box Binding Protein (TBP)** binds to the **TATA box** and marks site for RNA polymerase recruitment * **TFIID** recognizes the core promoter DNA sequence and TBP * Binding causes sharp bend dramatically marking spot * **TFIIF** finds RNA Pol II and ferries it to the initiation complex. * **TFIIH** joins and helps activate RNA Pol II * Has **helicase** activity to create transcription bubble * Has **kinase** activity that phosphorylates the **Carboxy Terminal Domain** (CTD) of RNA Pol II * **Activates polymerase** * Positions it directly over the DNA * As RNA pol II escapes the promoter it leaves the initiation complex behind

Question 117

Eukaryotic Transcription Elongation and Termination

Answer 117

* Once all initiation complex components and regulatory proteins in place, **polymerase escapes the promotor** * Makes several small abortive transcripts and starts **elongation** * Transcript is **terminated** when RNA Pol II reaches an **AAUAAA polyadenylation signal** * **GU-rich region** after polyadenylation signal causes polymerase to fall off DNA.

Question 118

mRNA Processing 7-methyl cap

Answer 118

* **Capping enzyme complex** adds a **7-methylguanosine triphosphate cap** to 5' end of primary transcript * Connected via a **5'-5' triphosphate linkage** * Helps protect from endonucleotytic degradation * Serves as marker that transcript is mRNA

Question 119

mRNA Processing Poly-A Tail

Answer 119

* AAUAAA **polyadenylation signal** recognized by **polyadnylation factors** * Recruit **endonuclease** to cut the RNA * Recruits **polyadenylate polymerase** to add poly A tail * Important for nuclear export * Length affects stability and half-life of mRNA

Question 120

mRNA Splicing

Answer 120

Spliceosome removes introns and joins exons snRNAs complex with proteins to form **small nuclear ribonucleoprotein particles (snRNPs)** * _Intron Splice Sites_ * 5' consensus GU **⇒ 5' splice donor site** * 3' consensus AG **⇒ 3' splice acceptor site** * conserved adenosine towards 3' end **⇒ branch site** * _Process:_ 1. snRNP bind the ends of the intron and the branch site A to loop out the intron 2. 2' OH from branch point A nuc. attacks G at 5' end creating a lariat 3. Free 3' OH of exon 1 attacks 5'-P at splice acceptor site joining exons 1 and 2

Question 121

tRNA Processing

Answer 121

* 5' leader sequence cleaved by RNase * 14 nucleotide intron removed by endonuclease * Uracil residues at 3' end replaced by "CCA" * Certain bases modified

Question 122

Operons

Answer 122

**A region comprised of several structural genes controlled by a common promoter and regulated by a common operator.** An **operator** is a segment of DNA that binds a **repressor** which regulates gene expression.

Question 123

LAC Operon Structural Genes

Answer 123

* **β-Galactosidase [Lac Z]** ⇒ intracellular enzyme that cleaves lactose into glucose and galactose * **β-Galactosidase permease [Lac Y]** ⇒ membrane-bound transport that mediates lactose entry into cells * **β-Galactosidase transacetylase [Lac A]** ⇒ transfers an acetyl group from acetyl-CoA to galactose

Question 124

LAC Operon Regulation

Answer 124

* **Lactose only ⇒ Lac operon induced (Positive regulation)** * Lactose → **allolactose** * Allolactose binds **repressor protein** ⇒ inactivates * Repressor **leaves operator** * When glucose absent, **adenylate cyclase** active ⇒ makes **cAMP** * cAMP binds **catabolite activator protein (CAP)** activating it * CAP binds **CAP binding site** causing ↑ transcription * **Glucose only ⇒ Lac operon repressed (Negative regulation)** * **Repressor bound to operator** site _downstream_ of promotor region * Prevents RNA Pol from binding to promotor * **Transcription blocked** * **Glucose + Lactose ⇒ derepressed state** * Lactose → allolactose ⇒ repressor * Repressor leaves operator * Transcription on but negligible since RNA Pol cannot efficiently initiate transcription without active CAP protein * Basal levels of gene expression

Question 125

Trp Operon Structure

Answer 125

* **Transcriptional control region** ⇒ operator within the promoter region * Unlinked **Trp R gene** codes for repressor protein * Not part of operon * 5 structural genes involved in last steps of tryptophan biosynthesis * Contains a **leader peptide** with an **attenuator sequence** (in mRNA transcript)

Question 126

Trp Operon Regulation

Answer 126

Operon sensitive to ratio of charged to uncharged tRNA^trp. **Attenuation** allows fine control. **_Repressor/Operator Control_** * High [trp], **trp** combines with **trp R repressor protein** * Complex **binds operator site** * **Transcription blocked** * Tryptophan acts as a **corepressor** (negative control) **_Attenuation_** Leader peptide with **attenuation sequence** (contains 2 trp codons) can form two hairpin loops. * High [trp] ⇒ trp incorporated quickly ⇒ ribosome moves quickly and blocks site 2 ⇒ hairpin forms between sites 3 and 4 making a termination signal ⇒ kicks off RNA pol and premature transcript forms * Low [trp] ⇒ trp incorporated slowly ⇒ ribosome stalls ⇒ alternative hairpin forms between sites 2 and 3 ⇒ trancription continues ⇒ full transcript made

Question 127

Eukaryotic Regulation of Expression Overview

Answer 127

The main level of control is at the transcriptional level. **Cis-acting DNA elements recruit trans-acting factors to initiate, modulate, or repress gene transcription.** Eukaryotic systems use coordinated transcription control ⇒ one master transacting factor regulating multiple genes. Multiple levels of regulation exist outside of transcription. Ex. **alternative mRNA splicing, control of mRNA stability, and control of translational efficiency.**

Question 128

GAL4 Transcriptional Control

Answer 128

Structural genes: Gal 1, Gal 10, Gal 2, Gal 7, Mel 1 Regulatory genes (constitutively expressed): Gal 4, Gal 80, Gal 3 **GAL4** binds to the **upstream activating sequence (UAS)** of all 5 structural genes activating transcription. **_Galactose Absent:_** * **GAL80 binds GAL4** preventing it from binding the UAS ⇒ represses transcription **_Galactose Present:_** * **Galactose** binds **Gal 3** ⇒ conformational change ⇒ complex **binds Gal 80 in cytoplasm** * Causes an equilibrium shift that **recruits the intranuclear Gal 80** bound to Gal 4 into the cytoplasm * **Free Gal 4 binds UAS and stimulates transcription**

Question 129

Alternative Splicing

Answer 129

Results in a single gene coding for multiple proteins. * **Splicing silencers** (cis-acting) bind **splicing-repressor proteins** (trans-acting) to **reduce** the chance that nearby site will be used as a splice junction. * _Located in neighboring exon_ ⇒ **exonic splice silencer (ESS)** * _Located whtin the intron itself_ ⇒ **intronic splice silencers (ISS)** * **Splicing enhancers** (cis-acting) bind s**plicing-activator proteins** (trans-acting) to increase the chance that nearby site will be used as a splice junction. * _Located in neighboring exon_ ⇒ **exonic splice enhancer (ESE)** * _Located whtin the intron itself_ ⇒ **intronic splice enhancer (ISE)**

Question 130

RNA Editing

Answer 130

mRNA undergoes nucleotide changes before translation. Increases protein diversity without increasing gene pool. Substiution editing most common. Two major systems both involve deamination reactions: * **Cytidine** → uracil by cytidine deaminase * **Adenosine** → inosine by adneosine deaminase

Question 131

Apolipoprotein B

Answer 131

Example of RNA editing. In liver, full mRNA transcript translated ⇒ apoB100 In small intestines, editosome changes C → U creating a stop codon. Only first 48% of transcript translated ⇒ apoB48

Question 132

Iron Metabolism Regulation

Answer 132

Expression of transferrin and ferritin regulated by **iron response elements (IREs)** [cis-acting]. * IREs form stem-loop structure which can bind **iron response proteins (IRPs)** [trans-acting]. * **5' IRE** prevents transcription. * **3' IRE** allows transcription and stabilizes MRA. * [IRP] high when [iron] low. * **Transferrin mRNA has 3' IRE** * **Ferritin mRNA has 5' IRE** When [iron] low, [IRP] high ⇒ IRP binds IRE ⇒ transferrin activated and ferritin repressed. When [iron] high, [IRP] low ⇒ IRP does not bind IRE ⇒ ferritin expressed and transferrin mRNA degraded.

Question 133

RNA Interference | (RNAi)

Answer 133

**RNA molecules used to interfere with gene expression.** Via destruction of mRNA or inhibition of translation. Role in defending against parasitic nucleotide sequences i.e viruses and transposons. * **microRNAs** (miRNAs) transcribed and processed. * In cytoplasm, **Dicer** (*endonuclease*) processes miRNA to produce **short DS miRNA.** * **Guide/Antisense strand** of DS miRNA hybridizes with RNA-induced silencing complex (**RISC**) * Guide strand targets mRNA for degradation or prevents translation RNAi can also be induced by exogenously added **siRNA**.

Question 134

Epigenetics

Answer 134

Mitotically and/or meiotically heritable changes in gene function that cannot be explained by changes in DNA sequence. Ex. ATP-dependent chromatic remodeling by HATs and HDACs. DNA methylation

Question 135

DNA Methylation

Answer 135

**DNA hypermethylation silences genes.** Cytosine bases in CpG-rich regions of promoters methylated by **DNA methyltransferases (DNMTs).** Unmethylated CpGs often grouped into **CpG islands**, present in **5' regulatory regions** of most genes.

Question 136

Hunters Syndrome | (MPS II)

Answer 136

* Defective **iduronate 2-sulfatase** * **Removes sulfate** from **sulfated alpha-L-induronic acid** * GAG present in **heparan sulfate and dermatan sulfate** * **X-linked recessive** * Presentation: * Mild type: lives to adulthood * Severe type: intellectual decline and rapid disease progression, lose basic function skills by 6-8 y/o, terminal around 10-12 y/o

Question 137

Hurler Syndrome | (MPS I)

Answer 137

* Defect in **alpha-L-iduronidase** * **Hydrolysis** of **unsulfated alpha-L-iduronic acid** * Present in **heparan sulfate and dermatan sulfate** * Heparan sulfate: glucuronic acid & N-acetylglucosamine * Dermatan sulfate: L-iduronate & N-acetylglucosamine * **Autosomal recessive** * More severe than other subtypes (MPS IS and MPS IH/S)