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131 Cards in this Set

  • Front
  • Back

Allosteric enzyme

an enzyme whose activity is affected by other substances binding to it



these substances change the enzyme’s activity by altering the conformation(s) of its quaternary structure

Allosteric effector

a substance that modifies the behavior of an allosteric enzyme


allosteric inhibitor


allosteric activator

Aspartate transcarbamoylase (ATCase)

made up of two different types of subunits


Catalytic subunit: 6 subunits organized into 2 trimers (Aspartate)


Regulatory subunit: 6 subunits organized into 3 dimers (CTP, ATP)

Feedback Inhibition

The final product inhibits the first reaction in the series


Efficient control mechanism that allows a shutdown of the entire series of reactions when there is excess of the final product


Not limited to allosteric enzymes

Sigmoidal curve

indicates cooperative behavior of allosteric enzymes


In the presence of CTP (inhibitor), a higher [S] (Aspartate) is needed for the enzyme to achieve the same rate of reaction


In the presence of ATP (activator), the rate of the reaction increases even at low [S].

Homotropic effects

Allosteric interactions that occur when identical molecules are bound (substrate)

Heterotropic effect

Allosteric interactions that occur when different substances are bound (Inhibitor and Substrate)

Concerted model

The substrate binds the R (active) form


Shifts equilibrium from T form to R-form (more R-form is produced)


Shifting equilibrium is responsible for the observed allosteric effect


Binding of Inhibitor: Stabilize T form, shifts equilibrium to T-form, more substrate is needed to shift T-form to-R form (greater degree of cooperativity)


Binding of Activator: Removes free R form, Shift toward more R -form to re-establish equilibrium. Less need for S to shift equilibrium in favor of R-form (less cooperativity)



Distinguishing feature: The conformation of all the subunits change simultaneously (concerted change)

R (relaxed) conformation

active form; binds substrate tightly

T (tight or taut) conformation

inactive form; binds substrate less tightly

Sequential model

Binding one molecule of substrate to one subunit induces the other subunits to adopt the R state, which has a higher affinity for substrate


Binding one molecule of inhibitor to one subunit induces a change in the other subunit to a form that has a lower affinity for substrate



Distinguishing feature: binding of substrate induces conformational change from T form to R-form. A conformational change in one subunit makes the same conformational change easier in another subunit

Phosphorylation

a type of covalent modification


The side chain –OH group of serine, threonine and tyrosine can form phosphate esters


Source of phosphate group is ATP


Phosphorylation by ATP can convert an inactive precursor into an active enzyme, or reduce the activity of an enzyme


Common example : Na+-K+ pump (Na+/K+ ATPase) found in all animal cells

Sodium-Potassium pump

Transport mechanism that pumps 3 Na+ out of the cell, and 2K+ into the cell, against a concentration gradient


Phosphorylation of the sodium–potassium pump is involved in cycling the enzyme between the form that binds to sodium and the form that binds to potassium

Glycogen phosphorylase

Catalyzes breakdown of stored glycogen


Exists in two forms –


Phosphorylated glycogen phosphorylase a (more active)


Dephosphorylated glycogen phosphorylase b (less active).

Covalent control

Phosphorylation converts b-form to a-form.


The kinase that puts the phosphates on is controlled by epinephrine (adrenalin)

Non-covalent control

Activated allosterically (T –R)


Increase in AMP (signals energy demand). This relaxed form has similar enzymatic properties as the phosphorylated enzyme.


Inactivated (R – T)


increase in ATP concentration ( sufficient energy stores)


Glucose (insulin signals glucose availability)

Zymogens

Inactive form of an enzyme that can be irreversibly transformed into the active enzyme by cleavage of specific peptide bonds


Chymotrypsinogen, Trypsinogen (inactive) , Trypsin (active), Procaspases (inactive), Caspases (active)

Chymotrypsinogen

synthesized and stored in the pancreas


Activated to chymotrypsin in small intestines

Trypsinogen (inactive) , Trypsin (active)

Activated by enteropeptidase

Procaspases (inactive), Caspases (active)

apoptosis or programmed cell death (cell turnover)

Which amino acid residues on the enzyme are in the active sites ?

Covalently modified versions of specific side chains (labeling)


Serine protease: a class of proteolytic enzymes in which the hydroxyl group of serine plays an essential role in catalysis


Chymotrypsin is a serine protease (uses S195)


S195 is covalently linked to DIPF (enzyme inactivated)



Three residues important for catalysis - Serine-195, Histidine-57, and Aspartate 102


These residues are arranged close to each other at the active site

How do the critical amino acids catalyze the chymotrypsin reaction?

Stage 1 :


Serine oxygen acts as a nucleophile (nucleus-seeking substance), and attacks the carbonyl group of the peptide bond of the substrate


The amino group of the peptide hydrogen bonds to the imidazole portion of histidine


The carbon-nitrogen bond of the original peptide breaks leaving the acyl-enzyme intermediate


Stage 2 (Deacylation stage) :


Water becomes hydrogen-bonded to the histidine


Water oxygen (acts as a nucleophile) attacks the acyl-enzyme intermediate.


The bond between the serine oxygen and the carbonyl carbon breaks releasing the product with a carboxyl group where the original peptide bond used to be Enzyme is regenerated.

Cofactor/coenzyme

a non-protein substance that takes part in an enzymatic reaction. It is regenerated for further reaction


Metal ions- electron pair acceptors that can behave as coordination compounds. (Zn2+, Fe2+)


Organic compounds- many of which are vitamins or are metabolically related to vitamins

Nicotinamide adenine dinucleotide (NAD+)

Nicotinamide adenine dinucleotide (NAD+) is a coenzyme in many oxidation-reduction reactions in biology.


Its structure is composed of


Nicotinamide ring


Adenine ring


two sugar-phosphate groups linked together


In oxidation-reduction reactions:


Main function: electron transfer reactions


NAD+ is an oxidizing agent – it accepts electrons from other molecules and becomes reduced to NADH


NADH can be used as a reducing agent to donate electrons.


Nicotinamide ring is where reduction-oxidation occurs

Chymotrypsin catalyzed reactions

Fast phase occurs first: the peptide bond is broken and the first peptide is released. The other peptide is covalently linked to the enzyme transiently.


Slow stage : second peptide is released, enzyme regenerated.

How to identify the critical amino acids in the active site

Labeling reagents

Lipids

compounds that are part hydrophobic


component of membranes, egg yoke, human nervous system



Open-chain compounds with polar head groups, and long non-polar tail groups


Fatty acids, Triacylglycerols, Phosphoacylglycerols, Sphingolipids, Glycolipids


Fused-ring compounds


Steroids

Fatty acids

Carboxyl group at the polar end and a hydrocarbon chain at the non-polar chain (amphipathic)


Length of fatty acid plays a role in its chemical character


Usually contain an even number of carbons (can contain odd)


Fatty acids that contain only C-C bonds, are saturated


Fatty acids that contain C=C, are unsaturated: The double bond is nearly always cis (gives a kink) and rarely conjugated


Unsaturated fatty acids have lower melting points than their saturated counterparts; the greater the degree of unsaturation, the lower the melting point


Rarely found free in nature – they form parts of many commonly occurring lipids

Degree of unsaturation

refers to the number of double bonds. The superscript indicates the position of the double bonds. For example ∆9 refers to a double bond at the ninth carbon atom from the carboxyl end of the molecule

Triacylglycerols (Triglyceride)

An ester of glycerol with three fatty acids


Accumulate in adipose tissues (as stored fatty acids for metabolic energy)



Hydrolysis of :


Enzyme hydrolysis – lipases


Acid or Base (KOH, NaOH) catalyzed hydrolysis


Natural soaps are prepared by boiling triglycerides with NaOH, in a reaction called saponification (Latin, sapo, soap)

Phosphoacylglycerols (Phospholipids/Phosphoglycerides)

Phosphatidic acid: One alcohol group of glycerol is esterified by a phosphoric acid rather than by a carboxylic acid


Phosphoacylglycerols are the second most abundant group of naturally occurring lipids (found in plant and animal membranes)


All have long, non-polar hydrophobic tails, and polar hydrophilic heads (amphipathic)


Nature of the fatty acid can vary widely

Waxes

A complex mixture of esters of long-chain carboxylic acids and alcohols


Found as protective coatings for plants and animals


Plants – coat stems, leaves, fruits


Animals- furs, feathers, and skin


Myricyl cerotate – component of carnauba wax (Brazilian wax palm). Used in floor and automobile wax


Cetyl palmitate: component of spermaceti (wax produced by whales) Used in cosmetics


Sphingolipids

Contain a long-chain amino alcohol called sphingosine


Found in plants and animals


Ceramide: one fatty acid is linked to amino group of sphingosine by an amide bond


Sphingomyelin: Primary alcohol group esterified to phosphoric acid which is in turn esterified to choline (amino alcohol)


Abundant in cell membranes in nervous system


amphipathic


Sphingomyelin bares structural similarity to phospholipids

Glycolipids

a compound in which a carbohydrate is bound to an -OH of the lipid by a glycosidic bond


Found in cell membranes of nerve and brain cells


In most cases, the carbohydrate (sugar) is either glucose or galactose


Many glycolipids are derived from ceramides (resulting compound is a cerebroside)


Glycolipids with complex carbohydrate moiety that contains more than 3 sugars are known as gangliosides


Gangliosides are present in nerve tissues

Gangliosides

glycoprotein with a complex carbohydrate portion


Carbohydrate portion contains more than three sugars. One of them is always a sialic acid

Steroids

A group of lipids that have fused-ring structure of 3 six-membered rings (A, B, C) and 1 five-membered ring (D).


Examples: cholesterol, testosterone, progesterone


Cholesterol is a precursor of other steroids and Vitamin D. Found in biological (animal) membranes (not in prokaryotic membrane)

Biological Membranes

Cells are encased by membranes composed of bilayers of lipids and proteins. Eukaryotic cells have additional membrane-enclosed organelles (such as nuclei, mitochondria)


Membranes separate cell from external environment, role in transport into and out of cell, some enzymes depend on membrane environment for function


Composition of lipids in membranes is important for membrane properties


polar head groups are in contact with the aqueous environment


nonpolar tails are buried within the bilayer


the major force driving the formation of lipid bilayers is hydrophobic interaction


the arrangement of hydrocarbon tails in the interior can be rigid (if rich in saturated fatty acids) or fluid (if rich in unsaturated fatty acids)

Lipid Bilayers

Principal lipid component is phosphoglycerides


Glycolipids, cholesterol (animals), phytosterols (plants)


The polar surface of the bilayer contains charged groups


The hydrophobic tails lie in the interior of the bilayer



Arrangement of hydrocarbon interior determines bilayer fluidity


Saturated hydrocarbons, close packing, ordered, - leading to rigidity


Kink(s) in hydrocarbon chain = causes disorder in packing against other chains


This disorder causes greater fluidity in membranes with cis-double bonds compared with saturated fatty acid chains

Cholesterol

Presence of cholesterol reduces fluidity (enhances order and rigidity)


Fused ring is quite rigid


Stabilizes extended chain conformations of the hydrocarbon tails of fatty acids as a result of van der Waals interactions

Transition temperature

With heat, ordered bilayers become less ordered


Transition temperature (temperature at which it changes from gel to liquid crystals) is higher for more rigid and ordered membranes, and lower for less rigid and disordered membranes


Gel to liquid transition


Thickness decreases


Surface area increases


Mobility of the lipid chains increases dramatically

Membrane Proteins

Several proteins are attached to membranes


Transport proteins: help move substances in and out of cell


Receptor proteins: transfer of extracellular signals (carried by hormones, neurotransmitters) into cells


Types


Peripheral proteins: On the surface of the membrane.


Integral proteins: cross both sides of lipid bilayer


May be completely embedded



Certain proteins are anchored to biological membranes by lipid anchors.


Particularly common are the N-myristoyl and S-palmitoyl anchoring motifs


N-myristoylation always occurs at an N-terminal glycine residue


S-palmitoylation involves thioester linkages at cysteine residues

Fluid-Mosaic Model

Most widely accepted description of biological membranes


Lipid bilayer has proteins, glycolipids, and steroids such as cholesterol embedded in it


Proteins and a lipid bilayer exist side-by-side (mosaic) without covalent bonds between the proteins and the lipids


Proteins tend to have a specific orientation in the membrane, can move along the plane of the membrane

Liposomes

an enclosed phospholipid bilayer structure (artificial membrane).


This stable spherical structure can be prepared with a drug inside it and used to deliver that drug to the tissues


Gets the drug to a place where it is most effective

Functions of Membranes

Three important functions that take place in/on membranes


Transport – allow flow of substances into or out of cells


Catalysis – Enzymes (proteins) bound to membranes catalyze reactions


Receptors – membrane proteins bind specific biological substances that trigger biochemical responses in cells

Passive transport

substance moves from a region of higher concentration to one of lower concentration (driven by a concentration gradient). Cell does not use energy


Simple diffusion: a molecule or ion moves through an opening in membrane


Facilitated diffusion: a molecule or ion is carried across a membrane by a carrier/channel protein

Active transport

substance moves from a region of lower concentration to one of higher concentration (against a concentration gradient). Cell uses energy


Primary active transport: transport is linked to the hydrolysis of ATP or other high-energy molecule; for example, the Na+/K+ ion pump


Secondary active transport: driven by H+ gradient

Simple diffusion

Passive diffusion of small uncharged molecules (CO2, O2, N2) across membrane


Driven by a concentration gradient

Facilitated Diffusion

Movement of molecules using a carrier protein


Example: glucose passes through glucose permease (carrier protein) into erythrocytes


Driven by a concentration gradient, no energy is used

Primary Active transport

Movement of molecules against a concentration gradient


Directly linked to hydrolysis of high-energy yielding molecule (e.g. ATP)

Secondary Active Transport

Couple molecule transmembrane transport with an energy source


Lactose concentration is higher inside the bacterial cell (requires energy to move lactose into cell


Proton pumps: integral membrane proteins that create a hydrogen ion gradient across the membrane


Galactose permease allows H+ to flow through it into the cell. One H+ is transported in with a lactose (harness energy for co-transport).

Membrane Receptors

Binding of a biologically active substance to a receptor initiates an action within the cell


LDL: low density lipoprotein; principal carrier of cholesterol in the blood stream


LDL is a particle that consists of phosphoglycerides, cholesterol, proteins



1. LDL binds LDL receptor


2. Receptor + bound LDL = vesicle; pinched off into cell (endocytosis)


3. LDL is released, releases cholesterol to be used in the cell


4. Receptor protein is recycled back to cell surface


5. Oversupply of cholesterol inhibits synthesis of LDL receptor


6. Too few receptors – level of cholesterol in blood increases


Atherosclerosis – blocked arteries


Heart attacks, strokes

Lipid-Soluble Vitamins

Vitamins are divided into two classes: lipid-soluble and water-soluble


Lipid soluble vitamins are hydrophobic


A, D, E, K

Vitamin A (Retinol)

Vitamin A (retinol) found in animals


Formed from enzymatic cleavage of B-carotene (unsaturated hydrocarbon)


B-carotene is abundant in carrots and other vegetables (yellow)


Enzymatic oxidation of Retinol (Vitamin A) forms retinal


Vitamin A participates in the visual cycle in rod cells


the active molecule is retinal (vitamin A aldehyde)


retinal forms an imine with an -NH2 group of the protein opsin to form the visual pigment called rhodopsin


Rhodopsin absorbs light


Isomerization of the 11-cis double bond to the 11-trans double bond


All trans-retinal and opsin released (cannot bind opsin)


Electrical impulse is generated in the optic nerve and transmitted to brain to be processed as a visual event


Enzyme isomerase regenerates 11-cis form leading to formation of rhodopsin


Vitamin A deficiency: blindness, night blindness


Excess vitamin A: bone fragility

Vitamin D

A group of structurally related compounds that are involved in the regulation of calcium and phosphorus metabolism


The most abundant form in the circulatory system is vitamin D3.


Formed from action of UV light (from the sun) on cholesterol.


Vitamin D3 leads to synthesis of calcium-binding proteins, increase in absorption of dietary calcium in the intestines, and calcium uptake by bones


Deficiency – rickets: bones become soft, leading to skeletal deformities

Vitamin E

The most active form of vitamin E is a-tocopherol


Required (in rats) for reproduction and prevention of muscular dystrophy


Vitamin E is an antioxidant (reacts with oxidizing agents before they can attack other biomolecules)


Removes very reactive and highly dangerous free radicals (HOO• and ROO•)

Vitamin K

Important in blood-clotting process


Two carbonyl groups (polar groups) & long unsaturated hydrocarbon side made up of repeating isoprene units


Number of isoprene units determine type of Vitamin



Vitamin K has an important role in the blood-clotting process


Required to modify prothrombin (adds a carboxyl group to alter glutamate residues) and other proteins


Unmodified prothrombin does not bind calcium


Anticoagulants dicumarol and warfarin are Vitamin K antagonists

Prostaglandins

Derived from fatty acids, widely distributed in tissues


Have a five-membered ring. Differ from each other by positions of double bonds and oxygen-containing functional groups


The metabolic precursor is arachidonic acid (20 carbon atoms: 4 double bonds)


Functions: control of blood pressure, induction of inflammation


Aspirin, cortisone and other steroids inhibit synthesis of prostaglandins (anti-inflammatory )

Leukotrienes

Compounds also derived from arachidonic acid


Found in white blood cells (leukocytes)


Consists of 3 conjugated double bonds


An important property is constriction of smooth muscles, especially in the lungs


Synthesis of Leukotrienes triggered by allergic reactions (eg; to pollen)


Asthma attacks may result from Leukotriene-induced constriction


Drugs that inhibit leukotriene C synthesis or block leukotriene receptors are used to treat Asthma

Thromboxanes

Also derived from arachidonic acid


Contain cyclic ethers as part of their structure


Thromboxane A2 induces platelet aggregation and smooth muscle contraction

Why should we eat more Salmon?

Omega-3 fats found in fish oils inhibit formation of certain prostaglandins and thromboxane A


Lower tendency for platelet aggregation, and a lower potential for artery damage

Nucleic acids

are macromolecules formed by the polymerization of nucleotides


Types of Nucleic acids


Ribonucleic Acid (RNA)


Deoxyribonucleic Acid (DNA)

Nucleotides

consist of three parts (covalently linked):


a nitrogenous base derived from purine or pyrimidine (nucleobases)


a sugar, either D-ribose or 2-deoxy-D-ribose


phosphoric acid


Nucleotide= Nucleoside + phosphoric acid


(phosphoric acid esterified to a hydroxyl group of sugar, example 5’-OH)


Name based on parent nucleoside, with a suffix, “monophosphate”, “diphosphate”, triphosphate (example: adenosine 5’-monophosphate)

Levels of NA Structure

Primary structure: the order of bases in the polynucleotide sequence ( the order of bases specifies the genetic code)


Secondary structure: the three-dimensional conformation of the polynucleotide backbone


Tertiary structure: supercoiling of the molecule


Quaternary structure: interaction between nucleic acids and other macromolecules to form complexes (ex: proteins)


Pyrimidine and Purine Bases

Pyrimidine:


Cytosine, Thymine, Uracil


Purine:


Adenine, Guanine

Nucleoside

Base + sugar

Primary structure of DNA

Backbone consist of alternating units of 2-deoxy-D-ribose and phosphate


3’-OH of one sugar is joined to the 5’-OH of the next sugar by a phosphodiester bond (repeating linkage is 3’, 5’-phosphodiester bond)


Base sequence is read from the 5’ end (has a phosphate group) to the 3’ end (free hydroxyl group).


Single letter A,G,C, and T

DNA – Secondary Structure

The three dimensional conformation of the backbone


Double helix: two polynucleotide chains wrapped around each other


coiled in a right-handed manner


antiparallel


Proposed by James Watson & Francis Crick, in 1953


Right and Left handed helices

Right- handed helices: helix winds upwards in the direction in which the fingers of the right hand curl when the thumb is pointing upward


Left-handed helices: helix winds upward in the direction in which the fingers of the left hand curl when the thumb is pointing upward

Base Pairing

A major factor stabilizing the double helix is complimentary base pairing by hydrogen bonding between T-A and between C-G


T-A base pair has 2 hydrogen bonds


G-C base pair has 3 hydrogen bonds

Other conformations (forms) of DNA

B-DNA


considered the physiological form, a right-handed helix, diameter 11Å, 10 base pairs per turn (34Å) of the helix


A-DNA


a right-handed helix, but thicker than B-DNA, 11 base pairs per turn of the helix, base pairs are not perpendicular to helix axis, lie at an angle of ~ 20o , has not been found in vivo


Z-DNA


a left-handed double helix, usually found in purine-pyrimidine alternating sequences (CGCGCG), may play a role in gene expression

Tertiary structure (Prokaryotic DNA)

Prokaryotic DNA is Circular: a type of double-stranded DNA in which the 5’ and 3’ ends of each stand are joined by phosphodiester bonds


Supercoiling

Supercoiling (Prokaryotic)

Further coiling and twisting of DNA helix.


Positive supercoil: overwound circular DNA, have more than the normal number of turns of a helix


Negative supercoil: underwound circular DNA, have fewer than the normal number of turns of a helix

Supercoiling in Eukaryotic DNA

More complicated than prokaryotes


Eukaryotic DNA is bound to a number of proteins


Chromatin: a complex of DNA and protein found in eukaryotic nuclei. Resembles “beads on a string”


Histones: the principal proteins in chromatin.


Basic proteins (H1, H2A, H2B, H3, H4)


Each bead is a nucleosome

Nucleosome

a globular structure in chromatin in which DNA is wrapped around an aggregate of histone molecules

Denaturation of DNA

Disruption of secondary structure


commonly by heat (melting)


Strands unwind, then separate to form single strands


Midpoint of transition (melting) curve = Tm


the higher the % G-C, the higher the Tm (why?)


Renaturation (annealing) is possible on slow cooling

RNA

consist of long, unbranched chains of nucleotides joined by phosphodiester bonds between the 3’-OH of one sugar and the 5’-OH of the next


the sugar unit is D-ribose (it is 2-deoxy-D-ribose in DNA)


the pyrimidine bases are uracil and cytosine (they are thymine and cytosine in DNA)


in general, RNA is single stranded (DNA is double stranded)


RNA molecules are classified according to their structure and function

Messenger RNA (mRNA)

carries coded genetic information from DNA to ribosomes for the synthesis of proteins


single stranded


a complementary strand of mRNA is synthesized along one strand of an unwound DNA, starting from the 3’ end

Transfer RNA (tRNA)

Transports amino acids to site of protein synthesis


a single-stranded polynucleotide chain between 73-94 nucleotides


intramolecular hydrogen bonding occurs in tRNA (A—U and G—C )


Different types found in living cells, frequently several tRNAs for each amino acid (carried at its 3’ end)

Ribosomal RNA (rRNA)

found in ribosomes, the site of protein synthesis


only a few types of rRNA exist in cells. Ribosomes consist of 60 to 65% rRNA and 35 to 40% protein

Small nuclear RNA (snRNA) is a recently discovered RNA

Found in nucleus of eukaryotes


Small (100-200 nucleotides long)


Associates with proteins to form small nuclear ribonucleoprotein particles (snRNPs)


snRNPs help with processing of initial mRNA transcribed from DNA

Micro RNA (miRNA)

small (~ 22 bp) non-coding RNA molecule found in plants, animals and some viruses


functions in RNA silencing and post-transcriptional regulation of gene expression.


miRNAs function by base-pairing with complementary sequences within mRNA molecules silencing these molecules

Information Transfer in Cells

Replication = process of duplicating DNA


Transcription = process of formatting RNA on a DNA template


Translation = process of protein synthesis in which the amino acid sequence of the protein reflects the sequence of the bases in the gene that codes for that protein



Information encoded in the nucleotide sequence of DNA is transcribed through synthesis of an RNA molecule


RNA sequence is determined by the DNA sequence


Sequence of RNA is read by protein synthesis machinery, and translated into amino acids in proteins

Central dogma of biology

DNA—RNA —Protein

Semiconservative Replication

Replication involves separation of the two original strands and synthesis of two new daughter strands using the original strands as templates


each daughter strand contains one strand from the original DNA, and one newly synthesized strand

Challenges in replication

1. Separating the two DNA strands


Strands must be unwound


Protection of single strands from nucleases


2. Synthesizing DNA from 5’ to 3’ end


two antiparallel strands must be synthesized in the same direction on antiparallel templates


3. Guarding against errors in replication


Correct base is added to the growing strand

In which direction does replication go?



Origin or Replication



Replication Fork

Replication is bidirectional


Origin of replication: the point at which the DNA double helix begins to unwind at the start of replication


Replication fork: the point(s) at which new DNA strands are formed


Prokaryotes: 1 origin of replication, 2 replication forks


Eukaryotes: Several origins of replication; 2 replication forks at each origin

Addition of a nucleotide to a growing DNA chain

DNA synthesis occurs in the 5’ to 3’ direction from the perspective of the growing chain


The 3'-hydroxyl group at the end of the growing DNA chain is a nucleophile. It attacks the phosphorus adjacent to the sugar in the nucleotide which is added to the growing chain. Pyrophosphate is eliminated, and a new phosphodiester bond is formed.

Semi-discontinuous model for DNA replication

DNA synthesis occurs in the 5’ to 3’ direction (from the perspective of the growing chain)


The leading strand is synthesized continuously in the 5’ -> 3’ direction toward the replication fork


The lagging strand is synthesized semi-discontinuously (Okazaki fragments) also in the 5’ -> 3’ direction, but away from the replication fork


The lagging strand fragments are joined by the enzyme DNA ligase

Requirements for DNA synthesis

All four deoxyribonucleotide triphosphates: deoxythymidine triphosphate (dTTP), deoxyadenosine triphosphate (dATP), deoxyguanosine triphosphate (dGTP), deoxycytidine triphosphate (dCTP), magnesium ions (Mg2+)


DNA (template) Primer: a short RNA strand to which the growing polynucleotide chain is covalently attached in the early stages of replication


Multiprotein complex called replisome (13 proteins in bacteria)


DNA polymerase: the enzyme that catalyzes the successive addition of each nucleotide to the growing DNA chain


There are at least five types of DNA polymerases (Pol) in E coli


Pol II, Pol IV, Pol V: repair enzymes

Proteins required for DNA replication

Helicase: unwinds DNA duplex


DNA gyrase: puts negative supercoils ahead of replication fork


Single-strand binding protein (SSB): protects single strand DNA from nucleases


Primase: copies a short stretch of DNA template strand to produce RNA primer sequence


DNA pol III: Each half of the replicative polymerase dimer is bound to its template strand by a β-subunit sliding clamp.


DNA pol I and DNA ligase act downstream on the lagging strand to remove RNA primers, replace them with DNA, and ligate the Okazaki fragments

3 essential activities of E. coli DNA polymerase I

1. 5' to 3' polymerization of DNA - makes phosphodiester bonds to create DNA strands


2. 3' to 5' exonuclease - proofreading function to remove nucleotide errors arising during replication


3. 5' to 3' exonuclease - to remove RNA primers

Proofreading

the removal of incorrect nucleotides immediately after they are added to the growing DNA during replication


DNA replication takes place only once each generation in each cell


Errors in replication (mutations) can be lethal to organisms


Errors in replication occur spontaneously only once in every 10^9 to 10^10 base pairs


Errors in hydrogen bonding occur once in every 10^4 to 10^5 base pairs


Pol I has proofreading, and repair functions. It can remove an incorrect nucleotide, and add the correct nucleotide

Mutations in DNA

Common mutagens: UV light, radioactivity, some chemical agents


UV irradiation causes dimerization of adjacent thymine bases - a cyclobutyl ring is formed between carbons 5 and 6 of the pyrimidine rings.


Normal base pairing is disrupted by the presence of such dimers.


The normal shape of the DNA is distorted

Oxidation Damage

Oxygen radicals, in the presence of metal ions such as Fe2+, can destroy the sugar rings in DNA, break the strand

Nick translation

DNA Polymerase I can fill in the gap after removing the nucleotides


DNA polymerase I can remove up to 10 nucleotides from a 3’-OH single strand nick

Mismatch repair

1. The newly synthesized DNA has a mismatch (G-T)


2. MutH, MutS, and MutI, link the mismatch with the nearest methylation site, which identifies the strand as the parental (correct) strand


3. Exonuclease I removes DNA from the strand between proteins


4. DNA polymerase III fills in the missing bases, DNA ligase seals the gap



Prokaryotes alter their DNA at certain locations by modifying bases with added methyl groups

Base-excision repair

A damaged base is removed from the sugar–phosphate backbone by DNA glycosylase, creating an AP (apurinic/apyrimidinic) site.


AP endonuclease removes the sugar and phosphate from the nucleotide


An excision exonuclease removes the AP site and several nucleotides.


DNA polymerase I fills the gap


DNA ligase seals the phosphodiester backbone

Nucleotide-excision repair

Commonly used to repair damage that deforms the DNA structure (pyrimidine dimers caused by UV light, chemicals )


ABC excinuclease binds to the region and cuts out a large piece of DNA, including the lesion.


DNA polymerase I and DNA ligase then resynthesize and seal the DNA.

Eukaryotic DNA Replication

A higher level of complexity


There are multiple origins of replication


More proteins and enzymes are involved


The timing must be controlled to that of cell division


In human cells, a few billions base pairs of DNA must be replicated once, and only once per cycle


Cell cycle times vary from less than 24 hours to hundreds of days

Initiation of DNA replication cycle in Eukaryotes

Origin recognition complex: multi-protein complex that binds DNA and serves as point of attachment for other proteins


Replication activator protein: an activation factor


Replication licensing factors: replication can not proceed until they are bound


Phosphorylation by cyclin and cyclin-dependent kinases


Initiates replication


Phosphorylated RAP and RLF are released (from ORC) and subsequently degraded.


G2 phase: DNA has been replicated


During mitosis DNA is separated into the daughter cells

Structure of the PCNA homotrimer

Proliferating cell nuclear antigen (PCNA) is part of Pol δ


It is the eukaryotic equivalent of the part of Pol III that functions as a sliding clamp (B-subunit).

Eukaryotic Replication Fork

DNA polymerase α: Primase activity and addition of first few nucleotides


After a few nucleotides are incorporated, DNA polymerase δ, with its associated proteins bind and do the majority of the synthesis.


Separate proteins (FEN-1 and RNase H1) degrade the RNA primers after replication.

Telomeres

Replication of linear DNA poses particular problems at the ends of the molecules


The ends of eukaryotic chromosomes have special structures called telomeres (series of repeated DNA sequences) which protect the end of the chromosome from degradation

Telomerase

an enzyme containing a section of RNA that is complementary to the telomere sequence. It uses this RNA as a primer to synthesize DNA at the ends of the chromosome


Removal of the primer shortens the DNA but it is now longer by one repeat unit


The telomerase extension cycle is repeated until there is an adequate number of DNA repeats for the end of the chromosome to survive

Comparison of DNA Replication in Prokaryotes and Eukaryotes

General Features of RNA synthesis

Transcription

Template strand (antisense, noncoding); DNA strand used as a template for the synthesis of RNA


RNA polymerase binds and transcribes the template strand


RNA polymerase moves from 3’ end to the 5’ on the DNA template strand


The mRNA is formed from the 5’ end to 3’ end


Nontemplate strand (coding/sense): The DNA strand that has the same sequence as the RNA that is synthesized


Transcription in Prokaryotes

Simplest of organisms contain a lot of DNA that is not transcribed


RNA polymerase needs to know which strand is the template strand


which part to transcribe


where first nucleotide of gene to be transcribed is located


Promoters: DNA sequence that provide direction for RNA polymerase (close to 3’ end of template strand)


Controls frequency with which a gene is transcribed

Elements of a bacterial promoter

Promoters have at least three components


Transcription start site (TSS): site used to initiate RNA synthesis


-10 region (Pribnow box): an essential part for transcription to occur


-35 region: Important in the control of RNA synthesis


UP element: a promoter element that is 40- 60 bases upstream of the transcription start site


Element: general term for a DNA sequence that is somehow important in controlling transcription

consensus sequences

Promoter regions in a number of prokaryotic genes contain base sequences that are common

Initiation and Elongation in Transcription

Factor independent termination (Intrinsic termination)

controlled by specific sequences


Two G-C rich regions (inverted repeats)


A-T rich region


Inverted repeats form a hairpin loop which destabilizes the association between RNA pol II and the DNA template


A-T rich region forms A-U base pairs which also destabilizes the association between RNA pol II and the DNA template, terminating transcription

Factor-dependent (Rho protein)

rho () protein binds a recognition site on mRNA, and moves along it toward the transcription bubble. When the Rho protein reaches the transcription bubble, at the termination site it causes dissociation (removal) of RNA pol from DNA template.

Transcription control in Prokaryotes

Enhancers and silencers: DNA sequences that transcription factors bind, to increase or reduce the level of transcription


Alternative σ factors: Expression of different σ-subunits that direct RNA pol to different promoters


Transcription attenuation: controls transcription after it has begun by adjusting the level of transcription based on the quantity of a related product


Operons: a group of genes that are controlled by the same promoter


Genes are only transcribed in the presence of an inducer (a small molecule)


Example induction of β-galactosidase (hydrolyzes lactose to galactose and glucose)


Catabolite repression in the Lac Operon

Catabolite repression: repression of the synthesis of lac proteins by glucose


Promoter has two regions


Binding site for RNA polymerase


RNA polymerase binding site overlaps with repressor binding site


Binding site for a regulatory protein, catabolite activator protein (CAP)


Binding of CAP depends on cAMP (“hunger signal”)


Cap-cAMP complex bound to CAP site , RNA pol I binds


Positive regulation


It takes the presence of lactose and the absence of glucose for the lac operon to be active


The trp operon in E.coli

Five proteins A-E make up four enzymes that catalyze synthesis of tryptophan


Trp R encodes a repressor protein that binds tryptophan


High levels of tryptophan, repression occurs


Low levels of tryptophan, five proteins are synthesized

Transcription Attenuation

Hairpin loops: Secondary structures formed in mRNA that are responsible for termination of transcription


1•2 pause structure: hairpin loop which forms and causes RNA polymerase to pause


3•4 terminator: hairpin loop which forms and causes premature release of the RNA transcript


2•3 antiterminator: hairpin loop which forms allowing transcription to continue

Attenuation mechanism in trp operon

When trp levels are high, ribosome passes over the trp codons quickly, Pause structure forms causing premature abortion of the transcript as the terminator loop is allowed to form.


When tryptophan levels are low, the ribosome stalls at the trp codons, allowing the antiterminator loop to form, and transcription continues


Transcription in Eukaryotes

More complex


Three primary RNA polymerases (each recognizes a different set of promoters)


RNA polymerase I: transcribes most ribosomal RNA (rRNA)


RNA polymerase II: transcribes messenger RNA (mRNA)


RNA polymerase III: transcribes transfer RNA (tRNA)


Numerous protein factors control transcription initiation


No operons - Genes to be transcribed depend on transcription factors


Elongation involves the addition of the 5'–phosphate of ribonucleotides to the 3'–OH of the elongating RNA (pyrophosphate is release)


Specific termination signals terminate transcription.

Pol II Promoters

Upstream elements: specific proteins bind this region to activate (enhancers) or suppress (silencers) transcription


TATA box: located ~25 – 100 bases before the transcription start site. Has consensus sequence TATAAT or TATAAA


Necessary for transcription in some genes, Orients the RNA polymerase correctly in others


Initiator element (lnr): includes TSS (+1) and surrounding sequences


Downstream element: a region for possible regulation (not as common as upstream elements)

Transcription factor

any protein that is not a subunit of RNA polymerase, but can regulate transcription

Sequence of events in Pol II transcription

A transcription factor (TFIID) binds the TATA box – first step in assembly of the pre-initiation complex


Poll II is phosphorylated before transcription starts.

Regulation of Transcription

DNA looping brings enhancers (or silencers) into contact with transcription factors and polymerase to activate or suppress transcription

Response Elements

short sequences of DNA within a promoter region that are able to bind specific transcription factors (produced under certain cell conditions) and regulate transcription.


Usually located in the promoter region of different genes, but can all be activated by the same stimuli, to produce a coordinated response


Heat shock response element (HSE) is present in heat shock protein genes. In response to high temperature, the heat shock transcription factor (HSTF) will interact with HSE, to activate the transcription of heat shock proteins (Hsp70, Hsp90)

Comparing Eu and Pro RNA

Zinc Finger motif

arises from zinc interacting with two closely spaced cysteines and two closely spaced histidines


Zinc finger proteins follow the major groove of DNA

Basic-Region Leucine Zipper Motif

A 30-amino acid segment with a periodic repetition of leucine residues at every seventh position


Other regions of the protein are rich in lysine and arginine residues

Posttranscriptional Modification of Eukaryotic mRNA

1. 5' end capping: Addition of a N-methylated guanine group to the 5’ end.


- Capping protects RNA from exonucleases.


2. Polyadenylation: A long sequence of adenosine residues (polyadenylate “tail”) that is usually100-200 nucleotides long, is added to the 3’ end of the mRNA


- polyadenylation protects the mRNA from nucleases and phosphatases


3. RNA splicing: the process of removing introns (non-coding sequences).


- RNA is transcribed from genes that contain introns which must be removed before RNA becomes biologically active


Capping of 5’-end of mRNA

Capping of the 5’ end with a methylated guanylate residue, bonded to the next residue by a 5’ -> 5’ triphosphate.


The 2’hydroxyl group of the neighboring ribose sugar is also frequently methylated (sometimes next neighbor as well)

The splicing reaction

Specific sequences make up the splice sites: GU at 5’ end of intron, AG at 3’ end of intron, Adenine (A) in the branch site


Advantage: A single gene can code for multiple proteins


- Tau ( found in brain of Alzheimer patients) has six isoforms


Abnormalities in the splicing process can lead to various disease states.


- Some forms of β-thalassemia are caused by mutations in the sequences required for intron recognition (leads abnormal processing of the β-globulin primary transcript)

Ribozymes

Proteins are not the only biological molecules with catalytic properties. Some RNAs, called ribozymes (ribonucleic enzymes), also catalyze certain reactions.


Some Ribozymes catalyze their own splicing (self-splicing)