WORKBOOK QUESTIONS

Name the three bacterial DNA polymerases and underline those which play a role in DNA replication.

** DNA POLYMERASE 1 ** DNA POLYMERASE 3 - DNA POLYMERASE 2

Biological importance of Pyrrole? And describe the electron distribution and acid-base property.

- Aromatic - Very weak acid Found in heme structure

Biological importance of Purine? And describe the electron distribution and acid-base property.

- Aromatic, amphoteric (Two rings - pyrimidine and imidazole fused together). - Important structural component of nucleic acids.

Biological importance of Indole? And describe the electron distribution and acid-base property.

- Aromatic, weak acid. - Structure found in amino acids like tryptophan.

Biological importance of pyridine? And describe the electron distribution and acid-base property.

- Aromatic, weak base. - Found in vitamin B6

What is the physiological, medical of Heroin? Give the acid-base properties as well.

- Base - No medical use.

What is the physiological, medical of Codeine? Give the acid-base properties as well.

- Can behave as a base. - Treatment of pain

Name three enzymes of bacterial DNA replication that cleave phosphodiester bonds between deoxyribonucleotides

- DNA POLYMERASE 1 - DNA POLYMERASE 3 - TOPOISOMERASE 2

Which enzyme of bacterial DNA replication removes the primers? Why is primer removal necessary?

- DNA POLYMERASE 1 removes the primers - Primer is made of RNA and not DNA

Which enzyme is necessary for the removal and the replacement of primers with newly synthesized DNA segments? Which enzyme activities does this enzyme possess?

- DNA Polymerase 1 - 3-5`exonuclease activity - 5`-3`Polymerisation

Which proteins are involved in prokaryotic DNA replication? Whats their function

- DNaA Protein = Recognizes origin sequence, opens duplex at specific sites in origin. - DNaC Protein = Unwinds DNA - Dam methylase = methylates GATC sequences at oriC

What is the similarities of DNA and RNA-Polymerase?

- Direction of synthesis 5->3` - Processivity - Hydrolysis of pyrophosphate - Mechanism of elongation

How can isoenzymes be distinguished form each other?

- Electrophoresis

Which structural change of bacterial DNA will the formation and movement of the replication bubble lead to? Which enzyme of replication can correct this structural change?

- It will create supercoils - DNA-gyrase can correct this structural change

What is the 4 major classes of lipoprotein particles?

- LDL - HDL - VLDL - Chylomicrons

What is the Functions of Nucleotides?

- Monomeric units of nucleic acids - Energy storages( ATP, GTP) - Components of coenzymes (CoA, NAD+, FAD).

What is the special of the RNA polymerase?

- No need for primer - No nuclease activity - pppA

Biological importance of piperidine? And describe the electron distribution and acid-base property.

- Non-aromatic, weak base - Structure can be found in morphine.

Biological importance of pyrrolidine? And describe the electron distribution and acid-base property.

- Not aromatic, weak base - When COOH binds it becomes Proline

What Is the function of Poly (A) tail?

- Protecting the pre-mRNA from degradation - Transport - Translational

What is the physiological, medical of morphine? Give the acid-base properties as well.

- Reduces pain at low doses, but narcotic effect at higher doses. - Amphoteric compound.

Differences between prokaryotic and the eukaryotic proteinsynthesis?

- Ribosome size and composition - initiatior tRNA - Initiation - structure of mRNA -elongation and termination - Energy costs

Isoenzymes can differ in?

- Sequence - Structure - Substrate affinity - Encoding genes

Isoenzymes catalyze the same reaction, but may differ in:

- Sequence - Structure - Subunit composition - Localisation - Substrate affinity (Km)

tRNA - function and structure

- Single strand of RNA folded into a precise three-dimensional structure. - It delivers amino acids to the site of proteins synthesis during translation.

What is the general rules of regulation? GIVE 4 EXAMPLES

- The first step of the reaction - pathway are mostly regulated steps. - The committed step of a pathway is always regulated - The regulated enzyme in most cases catalyses an "irreversible reaction"

List the mechanism responsible for maintaining the accuracy for protein synthesis.

- Wobble in codon-anticodon pairing - proofreading activity of aminoacyl-tRNA syntheases - spesific interactions between tRNA`s and aminoacyl-tRNA synthetases - codon-anticodon pairing check on the ribosome.

What can amino acid sequenve be used for?

- classification among protein types (function) - preparing antibodies spesific to the protein

Explain the smilitaries of DNA polymerase and RNA polymerase.!

- direction of synthesis 5´->3´ - mechanism of elongation - hydrolysis of pyrophosphate - processivity

Name 2 small RNA´s and their functions.

- miRNA = Silence genes by binding to mRNAs and can prevent transcription. - siRNA= Degradation of mRNA or inhibition of translation.

What is special with RNA polymerase?

- no need for primer - no nuclease activity - template: one strand of DNA, certain genes

List the proteins necessary for the unwinding of the DNA duplex during the initiation of bacterial DNA replication. Indicate the precise function and the ATP requirement of each listed protein.

-Helicase DNA B - - DNA A - DNA C

Which chemical bonds does the DNA contain? 1) Within the strand, between the nucleotides? 2) Within the strand, between the base and the deoxyribose? 3) Between the nucleotides or the two strands?

1 = Phosphodiester bond 2 = B-N glycosidic bond 3) Hydrogen Bond

Give a summary of cholesterol biosynthesis

1) 3 acetates condensate to form mevalonate 2) Mevalonate converts into 5-C isoprene 3) 6x Isoprenes polymerize and forma the «30-C Linear squalene» 4) squalene cyclizes to form the 4 rings that are modified to produce CHOLESTEROL

List the 5 steps of proteinsynthesis

1) Activation of aminoacids 2) initiation 3) elongation 4) termination and ribosome recylcing 5) folding and posttranslational processing

Transport across membranes, explain the concentration and electrochemical dependence:

1) C-Dependent= the solute moves toward equilibrium across the membrane 2) electrochemical dependence= the solute moves toward charge equilibrium across the membrane.

tRNA processing - - tRNA is produced by RNA Polymerase 3

1. 5´and 3´cleavage 2. Base modification - ex. methylation 3. "CCA-addition" Splicing

Describe the steps of bacterial DNA repair. (BASE-EXCISION REPAIR) = Removes abnormal bases f. ex hypoxanthine, xanthine)

1. Localisation of the DNA damage: DNA Glyosylase 2. Cleavage of the DNA strand that contains the damage: AP ENDONUCLEASE 3. Removal of a DNA fragment that contains the damage: DNA polymerase 1 4. Replacing the removed DNA: DNA polymerase 1 5. Sealing the nick: DNA ligase.

Describe the steps and proteins of bacterial DNA repair (MISMATCH REPAIR),,, ex. G-T or A-C

1. Localisation:Mut L and Mut S proteins. 2. Cleavage of the DNA strand that contains the damage: Mut H (endonuclease) 3. Removal of a DNA fragment that contains the damage by exonuclease action 3-5 and 5-3. 4. Replacing the removed DNA: DNA polymerase 3 5. Sealing the DNA-nick: DNA ligase

Describe the steps of bacterial DNA repair (Nucleotide-Excision repair) = removes DNA lesions that cause large distortions in the helical structure of the DNA.

1. localization of the DNA Damage: ABC-Exinuclease 2. Cleavage of the DNA strand that contains the damage: ABC Exinuclease 3. Removal of DNA fragment that contains the damage: DNA Helicase 4. replacing the removed DNA: DNA polymerase 1 5. Sealing the nick: DNA ligase

List the 4 mechanism of translation Regulation in proteinsynthesis

1. phosphorylation of translation initation factors 2. disruption of elF4E and elF4GF interactions 3. gene silencing 4. translational repressors

mRNA - function and structure

= carries the genetic code from DNA in a cell nucleus to ribosomes.

5- Cap synthesis and Poly (A) tail formation. Function?

A 5' cap (7 Methylo guanosine) is added to the beginning of the RNA transcript, and a 3' poly-A tail (phosphate group) is added to the end for protects the degradation of mRNA and helps the Ribosome to bind to the mRNA.

What is the role of the following enzymatic activities of DNA polymerase I during bacterial DNA replication? a) 3'-5' exonuclease activity, b) 5'-3' exonuclease activity.

A = proof reading B = Primer removal

Name that enzyme of bacterial DNA replication which: a) creates phosphodiester bonds between ribonucleotides b) cleaves phosphodiester bonds between ribonucleotides.

A) DNA PRIMASE B) DNA POLYMERASE 1

Name the proteins that function as endonucleases in the following bacterial DNA repair processes. a) mismatch repair, b) base-excision repair, c) nucleotide-excision repair

A= MUT H B= AP ENDONUCLEASE C= ABC EXINUCLEASE

Name those proteins that detect and localize the DNA alterations in the following bacterial repair processes. a) mismatch repair, b) base-excision repair, c) nucleotide-excision repair

A= MUT L AND MUT S B= DNA GLYCOSYLASE C= ABC EXINUCLEASE

DnaA protein function

ATPase enzyme that induces the denaturation of the DNA Unwinding region in the origin of replication (OriC)

DnaC protein function

ATPase enzyme that loads helices enzyme onto the separated DNA strands in the denatured region.

What is the function of tRNA aminoacid arm and D arm and Anticodon arm?

Aminoacidnarm= binds spesific aminoacids by ester bond Anticodon arm= interacts with the mRNA codon D arm= folding of tRNA

Biological importance of Imidazole? And describe the electron distribution and acid-base property.

Aromatic, amphoteric - Structure found in histidine.

Biological importance of Pyrimidine? And describe the electron distribution and acid-base property.

Aromatic, weak base. - found in vitamin B1

Medical function of Atropine?

Atropine is used in medical practice as a sulphate salt.

Explain briefly why we can say that bacterial DNA replication is semidiscontinuous.

Because one of the strand is being synthesized continuously, while the other strand is synthesized discontinuously by small Okazaki fragments.

D-Glyceraldehyde can be reduced to glycerol. why?

Because there is no asymmetry center in Glycerol.

What is the direction of DNA synthesis along the separated template strands? How do we name the newly synthesized DNA strands?

Both directions - Lagging strand.

Starting from the origin, which direction will the bacterial DNA synthesis proceed? Out of the two strands of the separated DNA duplex which strand will serve as the template of replication?

Both, Bidirectional.

Characterize the structure of sphingolipids. (Building blocks and bond types): what is their biological role?

Building block: fatty acid, sugar or phosphoric acid - sphingosine Bond type; glycosidic bond, amide and ester bond Biological role; main constituent of cell membrane, signalling

What is cofactors?

COENZYME (organic) or METAL ION (inorganic)

Give two examples of Cycloxygenase enzymes

COX 1 and COX 2

What is the function of mRNA in eukaryotic cell. And explain the 5`cap and Poly A- Tail.

Carries the genetic code from DNA nucleus to ribosomes 5-Cap; binds to the mRNA to the ribosome and protects mRNA from ribonuclease Poly A tail; requires endonuclease and Pol A polymerase and protects mRNA from enzymatic destruction

Function of phosphatidylinositol?

Cell signalling.

Function of vitamin B12?

Coenzyme e.g methylation.

What is the role of DNA methylation in the process of bacterial mismatch repair? Name the bacterial DNA methylase enzyme.

DNA METHYLATION is the mechanism for strand discrimination during replication error correction on the unmethylated newly synthesized strand. - DAM METHYLASE

Name and give the function of those enzymes that are required to join the Okazaki fragments.

DNA POLYMERASE - fills the gap - DNA LIGASE - ligation

Which bacterial DNA polymerase has 5-3`exonuclease activity? What is the significance of this enzymatic activity?

DNA POLYMERASE 1 - It removes the RNA primers in prokaryotic replication and replacing the removed DNA fragment.

Name those enzymes of bacterial DNA replication that cleave hydrogen bonds.

DNA-A HELICASE

What is Isoenzymes? Give examples.

Different proteins, but they catalyse the same reaction - Hexokinase, Cyclooxygenase

In which process are DnaA, DnaB, and DnaC PROTEINS INVOLVED?

DnaA = RECOGNIZES THE ORI SEQUENCE DnaB= Unwinds the DNA DnaC = Required for DnaB binding at origin.

Function of DnaB protein?

DnaB protein (helicase) DNA UNDWINDING

What is the difference between the mechanism of action of endonucleases and exonucleases?

EXONUCLEASES: Enzymes able to hydrolase nucleic acids one by one from one end of the molecule by cleaving phosphodiester bonds either in 3`-5`or 5`-3`direction. ENDONUCLEASES: can hydrolase phosphodiester bonds at specific internal sites in a nucleic acid strand or molecule, reducing it to smaller and smaller fragments.

What is the function of DNA polymerase 1 in Prokaryotic DNA REPLICATION?

Enzyme that removes the RNA primers of the newly synthesized leading and lagging strands, leaving a nick (broken phosphodiester bond).

What is the function of DNA polymerase 3 in PROKARYOTIC DNA REPLICATION?

Enzyme that synthesizes the complementary new DNA strands, the leading and the lagging strand.

What is Primase? And what is its function in the Prokaryotic DNA replication?

Enzyme that synthesizes the primers (short, complementary single stranded segments) for the newly synthesized leading and lagging strand.

What is Helicase - what does it do in the prokaryotic DNA replication?

Enzyme that unwinds the DNA double Helix by disrupting hydrogen bonds. It requires ATP for its function.

Describe the exonuclease function.

Enzymes able to hydrolase nucleic acids one by one from one end by cleaving phosphodiester bond either in 3-5 or 5-3 direction on one strand.

Describe the function of endonuclease.

Enzymes able to hydrolase phosphodiester bonds at specific internal sites in a nucleic acid strand, reducing it to smaller and smaller fragments.

What does DNA ligase do in Prokaryotic DNA REPLICATION

Enzymes that uses ATP or NAD+ to seal the nick in the broken phosphodiester bonds.

Characterize the structure of waxes (building blocks, bond types), what is their biological role?

Esters of long carbon chain C20, monovalent alcohols and long chain fatty acids. - Ester bond - protective function on skin, and hair

Myoglobin consists of

Globin + heme (prostethic group)

Which bond(s) in α-D-glucopyranose must be broken to change its configuration to β-D-glucopyranose?

Glycosidic bond.

Composition of enzymes, what is a Holoenzyme?

HOLOENZYME = APOENZYME + COFACTOR: complete, catalytically active enzyme

Give the reaction of oleic acid and Bromine. What kind of reaction is this?

Halogen-addition

Compare the enzyme kinetics parameters of hexokinase 1 and hexokinase 4 enzymes.

Hexokinase 1 = - Occurs in every tissue except liver and pancreas. - Non-specific for glucose Hexokinase IV= - Occurs in liver and pancreas. - spesific for glucose - Not inhibited by Glucose 6-Phosphate

What is a histone? How can we affect the charge?

Histones contains basic Aminoacids which tightly binds to the DNA.

Function of vitamin K1?

Important in Blood clotting and bone buildimg.

Explain the term splicing.

In splicing, some sections of the RNA transcript (introns= Noncoding sequences, f. ex) are removed, and the remaining sections (exons) are stuck back together. Ex. Self-Splicing "G-Factor".

Where is cholesterol synthesized?

In the liver, and then exported as bile acids which is stored in the Gall bladder.

How does the use of NSAIDS influence the biosynthesis of prostaglandins, thromboxanes and leukotrienes?

Inhibits production of prostataglanding and Thromboxane, and indirectly increases production of Leukotrienes.

How does the initiation of replication work at the E.Coli?

Initiation starts a specific DNA-sequence called ORIGIN. Dna-A protein with ATP recognizes and binds to this ORI-sequence and «opens» the double-helix structure).

Which 3 major classes are the membrane proteins?

Integrate, peripheral and amphitropic

Function of Uridine diphospholgucose?

Intermediate in carbohydrate metabolism.

Function of vitamin D3

Is produced in the skin by UV Radiation and it regulates the metabolism of Ca2+ in the kidney

What is DNA replication?

Is the process by which a double-stranded DNA molecule is copied to produce two identical DNA molecules. Proceeds in 5->3 direction.

Where does the allosteric effector bind? By what type of bond does it bind?

It bind on the regulatory site by secondary interactions.

What is the characteristic structural feature of saponifiable lipids? What is its consequence?

It contains ester or amide bond which can be hydrolysed by acid or bases.

What is the term "turnover number of an enzyme"?

It is the maximal number of molecules of substrate converted to product per active site per unit of time.

Give a short definitin of dissociation constant (Kd)

Kd is the logand concentration where half of the binding sites are occupied

Which newly synthesized DNA strand is formed by Okazaki fragments? What do the Okazaki fragments consist of?

Lagging strand. It consist of a primer with a short piece of DNA

Which DNA strand will be cleaved by the endonuclease? What type of labelling allows differentiation between the two strands of DNA?

Methylated strand

What does a mismatch in the structure of the DNA mean and how does it affect the formation of secondary interactions between the deoxyribonucleotides?

Mismatches occur when the bases of the DNA strands are not complementary, which mean they don't form hydrogen bond with each other.

Explain the function of antibiotics

Molecules produced to kill microorganisms (prokaryotes or eukaryotes) e.g Streptomyocin

What does the term: enzyme inhibitor mean? what are the two main classes of inhibitors.

Molecules that interfere with catalysis, slows or stops enzymatic reactions. - reversible/ irreversible inhibitors.

What is the consequence of the difference between hemoglobin and myoglobin?

Myoglobin has higher affinity for oxygen than Hemoglobin. Which means it keeps the oxygen bounded even as the p02 decreases.

Function of NAD and FAD?

NAD+ = Transfer of 2H ATOM. (ENZYME = DEHYDROGENASES) FAD = Transfer of 1 or 2H atoms. (Enzyme = Dehydrogenase)

Name a P-type ATPase, briefly describe its mechanism of action, and indicate its biological significance.

Na+-K+ ATPase. For every ATP molecule the pump uses, 3 Na+-ions are exported and 2 K+ ions are imported. (Membrane potential)

By the addition of the enzyme how do the following change: • the value of ΔG(0)? • the value of ΔG? • the value of ΔG‡?

No change except for the ΔG‡-value = Ea will decrease.

Steps of bacterial DNA repair (DIRECT REPAIR). = DAMAGE ARE REPAIRED WITHOUT REMOVING A BASE OR NUCLEOTIDE.

O6-Methylguanine DNA methyltransferase.

Explain briefly why we can say that bacterial DNA replication is semiconservative.

One chain of the replicated DNA is the original strand, while the other is the newly synthesized strand.

Function of Porphines?

Plays a vital role in oxygen transport

What is the enzyme involved in the poly (A) tail?

Polyadenylate polymerase.

Structure of DNA?

Primary = order of nucleotides Secondary = double helix, antiparalell Secondary = superhelix or circular

Name the short RNA segments required for the function of bacterial DNA polymerases. Which enzyme of bacterial DNA replication is responsible for the synthesis of these short segments?

Primers - Made by Primase

What is the SSB (Single stranded binding proteins) function in the prokaryotic DNA replication?

Proteins that bind and stabilize the separated single strand of the DNA.

Show the equation of Poly (A) tail formation.

RNA + nATP --> RNA-(AMP)n + nPPi

Beside DNA repair which other DNA related process is this polymerase involved in, and what is the function of it in that other process?

Replication.

Explain the direct repair in bacterial DNA repair.

Several types of damage are repaired without removing a base or a nucleotide. Ex. O6-methylguanine DNA methyltransferase corrects alkylated Nucleotides - DNA photolyase corrects pyrimidine dimers

Describe the common structural features and the differences for D-Glucose and D-Fructose

Similarity: Both Monosaccharides are aldohexoses with same molecular formula. Difference: - They differ in the configuration around the 4.th carbon atom. - D-Glucose is an aldohexose with aldehydic functional group. - D- fructose is a ketohexose with a ketonic functional group.

Describe the common structural features and the differences for Maltose and Sucrose:

Similarity: Both are disaccharides. Difference: Maltose is a combination of Glucose and Glucose, while sucrose is a combination of glucose and fructose. - Sucrose is a non-reducing sugar and maltose is a reducing sugar.

What does the term "racemic mixture mean"? Characterize the optical activity

The 1:1 mixture of enantiomers is called racemic mixture. Optically inactive, because the activities of the two asymmetric molecules compensate each other

What kind of change in the structure of the enzyme is caused by the binding of the allosteric effector? How does this change appear in the enzyme function?

The allosteric regulation is a reversible change, which depends on the concentration of the effector and appears generally in short-term effect.

Why is the bacterial polymerase with the highest processivity required for this repair process?

The damage can be far away so we need the one with highest activity = Polymerase 3

Hydrolysis of the glycosidic bond - explain how it can be cleaved

The glycosidic bond cand be cleaved by acidic treatment. In living organisms the hydrolysis of Oligo and Polysaccharides is catalyzed by enzymes e.g amylase.

Explain the Bohr effect

The influence of pH and CO2 on the oxygen affinity of hemoglobin

Which newly synthesized DNA strand is formed by Okazaki fragments, and which enzymes are required for their synthesis?

The lagging strand is formed by Okazaki fragments. - DNA polymerase 1 and DNA primate is required for their synthesis

Proofreading = (3`-> 5`exonuclease activity). How does it work?

The proofreadings "active site" on the DNA polymerase slides back to the position of the mismatch and removes the nucleotide. DNA polymerase slides forward and continues with its polymerization.

Compare the UV absorption of NAD in oxidized and reduced form.

The reduced NADH absorbing at 340 nm, while the oxidized NAD+ dosent.

Give the reaction of stearic acid and sodium hydroxide. Compare the watersolubility for starting material and that of the product.

The «salt» end of the molecule is ionic and is water soluble

Function of vitamin A

To improve vision and keeping skin & healthy.

Name those four enzymes of bacterial DNA replication that create/seal phosphodiester bonds between deoxyribonucleotides.

Topoisomerase 2 DNA LIGASE

What is the functiom of TOPOISOMERASE 2 (Dna gyrase) in prokaryotic DNA replication?

Topoisomerases are enzymes that cleaves and resealing phosphodiester bonds either on one strand(Topoisomerase 1) or both strands (topoisomerase 2-ATP dependent) to relax the supercoiled DNA.

Exlpain GOUT

Uric acid accumulation that lead to deposit of sodium rate crystals in joints and capillaries causing inflammation (GOUT)

Why does this repair process require exonucleases with 3'→5' and 5'→3' activity?

We don't know on which side the damage is compared to the Hemimethylated GATC is.

Function og Spingomyelin?

Wraps around axon in nerve system.

What is prostethic group?

a cofactor which binds to the enzyme very strongly, even covalently (it can be both a coenzyme or a metal ion).

What is a domain?

a distinct polypeptide chain in the protein that has a specific function. (e.g. catalytic, regulatory, transmembrane, substrate binding, connecting domain)

function of cAMP

activates protein kinase A and signal transduction.

Function of Vitamin E?

antioxidant

Explain the pka activation

cAMP activates PKA, this is a part of the epinephrine signaling pathway

List the main Eukaryotic RNA types and which are producing them.

mRNA - Eukaryotic RNA polymerase 2 (alos snRNA) rRNA - Eukaryotic RNA Polymerase 1 and 3 tRNA - Eukaryotic RNA Polymerase 3

What is a apoenzyme?

the protein that remains after the removal of coenzymes or metal ions

rRNA -function and structure

within the ribosome, proofreading.

What does the high and low value of Km indicate?

•Low KM - great affinity for the substrate •High KM - low affinity for the substrate