Why is the 5' end of DNA a monophosphate?

Why is the 5' end of DNA a monophosphate?

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According to my textbook:

While the 5' end of a DNA strand is typically a monophosphate, the 5' end of an RNA molecule is typically a triphosphate.

Source: Biology: How Life Works, 3rd Edition

How do we know the 5' end of DNA a monophosphate? I understand that…

  • DNA and RNA synthesis cleaves nucleoside triphosphates into nucleoside monophosphates to form the sugar-phosphate backbone.
  • Necessarily, the first nucleotide will have three phosphates intact.

What I don't understand is why DNA doesn't have a triphosphate on the 5' end like RNA. How does that happen?

Base Pair

A base pair refers to two bases which form a "rung of the DNA ladder." A DNA nucleotide is made of a molecule of sugar, a molecule of phosphoric acid, and a molecule called a base. The bases are the "letters" that spell out the genetic code. In DNA, the code letters are A, T, G, and C, which stand for the chemicals adenine, thymine, guanine, and cytosine, respectively. In base pairing, adenine always pairs with thymine, and guanine always pairs with cytosine.

More: DNA is 'read' in a specific direction, just like letters and words in the English language are read from left to right. Each end of DNA molecule has a number. One end is referred to as 5' (five prime) and the other end is referred to as 3' (three prime). The 5' and 3' designations refer to the number of carbon atom in a deoxyribose sugar molecule to which a phosphate group bonds.

This slide shows how the carbons in the sugars are numbered, to help you determine which ends is 5', and which is 3'. Once you figure out the direction in which one strand is read, you automatically know the direction in which to read the other strand. This is because the two strands are also antiparallel (they run in opposite directions), as mentioned in the previous slide.

DNA Replication: Mechanisms of DNA Replication

DNA replication in eukaryotes is semiconservative, semi-discontinuous and bidirectional as compared to semiconservative, bidirectional and continuous in prokaryotes.

Image Courtesy :

DNA replication occurs during S-phase of cell cycle. It is a multistep complex process which requires over a dozen enzymes and protein factors. It begins at a particular spot called origin of replication or ori. Bacterial and viral DNA has a single origin of replication. It functions as a single replicating unit or replicon.

In eukaryotic DNA there are a number of origins of replication. It has several replicating segments or replicons, i.e., multirepliconic. In the absence of ori, replication will not occur. The requirement of a vector for recombinant DNA technology is to obtain origin of replication.

Replication of DNA is energetically highly expensive. The main enzyme of DNA repli­cation is DNA dependent DNA polymerase. DNA replication is quite rapid. The replication of DNA of E. coli with 4.6 x 10 6 bp requires 19 minutes.

On an average rate of polymer­ization of bases is 2000 bp per second in each direction. Replication requires abundant energy that comes from breakdown of triphosphates of deoxyribonucleotides.

Replication takes place as follows:

1. Activation of Deoxyribonucleotides:

Deoxyribo­nucleotides or deoxyribonucleoside monophosphates occur freely inside the nucleoplasm. They are of four types— deAMP (deoxyadenosine monophosphate), deGMP (deoxyguanosine monophosphate), deCMP (deoxycytidine monophosphate) and deTMP (deoxythymidine monophosphate). They are first phos- phorylated and changed to active forms which have three phosphate residues instead of one. Enzymes phosphorylase is required along with energy.

The phosphorylated nucleotides are deATP (deoxyadenosine triphosphate), deGTP (deoxyguanosine triphosphate), deCTP (deoxycytidine triph­osphate) and deTTP (deoxythymidine triphosphate). These triphosphates of bases serve dual purpose. They act as sub­strate as well as provide energy for polymerisation of nucleotides.

2. Exposure of DNA Strands:

Enzyme helicase (unwindase) acts over the Ori site and unzips (unwinds) the two strands of DNA by destroying hydrogen bonds. The separated strands are stabilised by means of single stranded binding proteins (SS BPs) or helix stabilising proteins. Un­winding creates tension in the uncoiled part by forming more supercoils. Tension is released by enzymes topoisomerases.

They cause nicking of and resealing the DNA strand. Along with topoisomerase, bacteria possess another enzyme called DNA gyrase which can introduce negative supercoils (older workers believed that gyrase functioned both for helicase and topoisomerase).

With the help of various enzymes both the strands of DNA become open for replication. However, whole of DNA does not open in one stretch due to very high energy requirement. The point of separation proceeds slowly towards both the directions. In each direction, it gives the appearance of Y-shaped structure called replication fork (Fig. 6.13 and 6.14).

3. RNA Primer:

It is essential for initiation of new DNA chains. RNA primer is a small strand of RNA which is synthesised at the 5′ end of new DNA strand with the help of DNA specific RNA polymerase enzyme called primase. RNA primer is formed on the free end of one strand and fork end of the other strand. Formation of RNA primer constitutes the initiation phase of DNA synthesis because without the presence of RNA primer, dna polymerases cannot add nucleotides.

A more complex enzyme called primo some is required in phage ф x 174 and some other prokaryotic systems. In eukaryotes, the function of primase is carried out by enzyme DNA polymerase α. It builds up

10 base RNA and 20-30 bases of DNA (Lewin, 2004). After start of nucleotide chain, RNA primer is removed and the gap is filled by DNA polymerase I in prokaryotes and DNA polymerase β in eukaryotes.

4. DNA Polymerases:

Prokaryotes have three major types of DNA synthesising en­zymes called DNA polymerases III, II and I. All of them add nucleotides in 5’—>3′ direction on 3′ —> 5′ stretch of parent strand. They also possess 3’—>5′ exo-nuclease activity. While DNA polymerase III is mainly involved in DNA replication (addition and polymerisation of new bases), polymerase I is major repair enzyme. Polymerase II is minor repair enzyme.

DNA polymerase I also has 5 —> 3 exonuclease activity. In eukaryotes five types of DNA polymerases are found— α, β, γ, δ, and ε, but the major three being α, δ and e. Polymerase 8 is involved in replication of leading strand. Polymerase may help in synthesis of lagging strand alongwith other roles. Polymerase α is largest and main enzyme of replication of DNA. All DNA polymerases have a configuration of gripping hand with thumb on one side, fingers on the other and the palm like concave catalytic site for combining template and base pairs.

5. Base Pairing:

The two separated DNA strands in the replication fork function as templates. Deoxyribonucleoside triphosphates come to lie opposite the nitrogen bases of exposed DNA templates — deTTP opposite A, deCTP opposite G, deATP opposite T and deGTP opposite C.

Nucleophilic attack separates a pyrophosphate (PPi) from the triphos­phate. Phosphodiester linkages are established. Hydrolysis of pyrophosphate by enzyme pyrophosphatase releases energy. Deoxyribouncleoside triphosphate → Deoxyribouncleoside monophosphate + PPi

The energy is used in establishing hydrogen bonds between the free nucleotides and nitrogen bases of templates.

6. Chain Formation:

It requires DNA polymerase III (Kornberg, 1956) in prokaryotes and polymerase δ /ε in eukaryotes. DNA polymerase III is a complex enzyme having seven subunits (a, β, ƍ, ƴ, €, θ, τ). In the presence of Mg 2+ , ATP (GTP), TPP and DNA polymerase III, the adjacent nucleotides found attached to nitrogen bases of each template DNA strand establish phosphodiester bonds and get linked to form replicated DNA strand.

As replication proceeds, new areas of parent DNA duplex unwind and separate so that replication proceeds rapidly from the place of origin towards the other end. RNA primer is removed and the gap filled with complementary nucleotides by means of DNA polymerase I. Because of sequential opening of DNA double chain and its replication to form two chains, DNA replication is also called zipper duplication.

However, DNA-polymerase can polymerise nucleotides only in 5’→ 3′ direction on 3′ —> 5′ strand because it adds them at the 3′ end. Since the two strands of DNA run in antiparallel directions, the two templates provide different ends for replication. Replication over the two templates thus proceeds in opposite directions. One strand with polarity У —> 5′ forms its complementary strand continuously because 3’end of the latter is always open for elongation.

It is called leading strand. Replication is discontinuous on the other template with polarity 5′ → 3 because only a short segment of DNA strand can be built in 5′ → 3 direction due to exposure of a small stretch of template at one time. Short segments of replicated DNA are called Okazaki fragments (= Okasaki segments Reiji Okazaki, 1968). Each of them has 1000- 2000 bp in prokaryotes and 100-200 bp in eukary­otes.

An RNA primer is also required every time a new Okazaki fragment is to be built. After replacing RNA primer with deoxyribonucleotides and their polymerisation, Okazaki frag­ments are joined together by means of enzyme, DNA ligase (Khorana, 1967). DNA strand built up of Okazaki fragments is called lagging strand.

As one strand grows continuously while the other strand is formed discontinuously, DNA replication is semi-discontinuous. Since replication proceeds bidirectionally from the origin of replication or ori, one parent strand will form a leading strand on one side and lagging strand on the other side. The reverse occurs on the parent strands of the other side. This helps in completing replication simultaneously in the whole replicon.

7. Proof-reading and DNA Repair:

A wrong base is sometimes introduced during replication. The frequency is one in ten thousand. DNA polymerase III is able to sense the same. It goes back, removes the wrong base, allows addition of proper base and then proceeds forward. However, even DNA polymerase III is unable to distinguish uracil from thymine so that it is often incorporated in place of thymine. Such a mismatching is corrected by means of a number of enzymes.

There is a separate repair mechanism for any damage caused to DNA due to mutation, UV exposure or mismatching that escapes proof-reading mechanism. A nick or break is caused by an endonuclease near the region of repair. DNA polymerase I (Komberg, 1969) removes the mismatched or wrong nucleotides if present and synthesises a correct replace­ment by using the intact strand as template. The newly formed segment is sealed by DNA ligase.

Phosphate Group Function

In Cellular Energy

One of the main functions of a phosphate group within cells is as an energy storage molecule. When a phosphate group is added to a molecule of adenosine, it becomes adenosine monophosphate, or AMP. This molecule is used in a number of biochemical reactions, and is heavily involved in both storing energy and as a second messenger in cellular signaling.

When you add another phosphate group, you get adenosine diphosphate (ADP). This molecule has an additional phosphate group bound to the first, and stores energy in this bond. This ADP molecule can accept another phosphate group, and become adenosine triphosphate. Commonly called ATP, this molecule can transfer the third phosphate group to a number of enzymes, activating them or imbuing energy to some process. You can see the recycling of phosphate groups between ADP and ATP in the diagram below.

Phosphate groups are one of the most important cellular components. Unfortunately for organisms, it is primarily based on a source of phosphorous atoms. This is the reason phosphorous is commonly a limiting nutrient. It is commonly a component of fertilizer for agricultural crops, which allows both the plants and the microorganisms in the soil to thrive.

Within DNA

A phosphate group is also a key component of life itself. A constituent of deoxyribonucleic acid is a number of individual phosphates. DNA is composed of individual units, called nucleotides. Each free nucleotide has two additional phosphate groups, which will be used in the reaction binding it to the DNA chain. The process can be seen in the following diagram.

Each nucleotide contains a nucleotide base (A,T,C, or G), a sugar (deoxyribose), and a phosphate group. The chain of DNA is formed by bonds between the phosphate group of one molecule to the sugar molecule of the next. These series of phosphodiester bonds become the sugar-phosphate backbone of the molecule. This is also true of RNA, but the sugar is different (ribose).

Cyclic AMP

Another major function of a phosphate group within biological systems is as part of the cellular messenger, cyclic adenosine monophosphate. Also known as cyclic AMP, or simply cAMP, this molecule is used in a number of signal transduction pathways. Signal transduction is the process of transmitting a chemical signal through the cellular membrane. It involves a number of proteins, and often a phosphorous group or two.

Typically, a signal transduction pathway starts by a chemical arriving at an integral membrane protein. These proteins cross the cellular membrane. When the protein is activated by the chemical, it changes shape slightly, activating another enzyme inside of the cell membrane. This enzyme, adenylate cyclase, uses the energy from two phosphate groups of an ATP molecule to produce a cAMP. These signal molecule then affect a number of other proteins, channels, and enzymes, leading to an overall cellular reaction. This use of a phosphate group (or many) is seen in many cellular communication channels.

Other Uses of a Phosphate Group

A phosphate group is also a component of the lipid bilayer which creates cellular membranes. Each phospholipid molecule within the bilayer has a phosphate group at the head of the molecule. The phosphate group is hydrophilic, attracting the head of the molecule towards water. The hydrophobic tails are collected together, forming a semi-permeable membrane which separates the contents of the cell from the outside.

A free phosphate group within the cytoplasm can also act as a buffer, attaching to strong acids or bases, and decreasing their effect on environment as a whole. This helps cells maintain an regular and consistent pH, and allows cellular processes to evolve.

Gene Probes: Concept, Labelling and Translation

The information needed to produce a gene probe may come from many sources but with the development and sophistication of genetic database, gaining this knowledge is usually one of the first stages.

In some cases it is possible to use related proteins from same gene family to gain information on the most useful DNA sequences.

Protein or DNA sequences that are similar but are from different species may also provide a starting point with which to produce a so called hetero­logous gene probe.

Also with the help of database, single stranded oligonucleotide probe can be synthesized chemically. This is done by computer controlled gene synthesizers that link dNTPs together on the basis of a desired sequence. Whatever way is used to obtain a probe but it should be assured that probe is unique, and is neither able to self-anneal nor is self-complimentary.

Where scant DNA information is available to prepare a gene probe it is possible in some cases to use the knowledge obtained form analysis of protein as from genetic code it is possible to predict the various DNA sequences which could code for the protein and thus then can synthesize oligonucleotide sequence chemically.

However, due to degeneracy of genetic code there could be more than one oligonucleotide for a given polypeptide. Ideally, sequence that is specific for a given gene and is not longer than 20 bases that contains as many tryptophan and methionine as possible is sufficient as these have unique codons and therefore, fewer possible base sequence that can code for that part of protein.

Labelling of Gene Probes:

An essential feature of gene probe is that it can be visualized by some means. Therefore, labeling of probe is a must and is done by two methods, traditionally by using radioactive labels and by non-radioactive labels. Mostly used radioactive labels are phospho­rous 32 ( 32 P), sulphur 35 ( 35 S) and tritium ( 3 H) which are detected by process of autoradiography.

Non-radioactive probes, although less sensitive than radioactive probes but are safe to use. The labelling sys­tems are termed either direct or indi­rect. Direct labelling allows an enzyme reporter such as alkaline phosphatase to couple directly to DNA. Indirect labelling method which is more popu­lar involves binding of nucleotide that has label attached, e.g., Biotin, fluorescein and digoxigenin.

These molecules are covalently linked to nucleotides. Specific binding proteins may then be used as a bridge between nucleotide and reporter protein such as enzyme. For example biotin incorporated into DNA molecule is recognized with a very high affinity by Streptavidin. This may be in turn is coupled with enzyme alkaline phosphatase which is able to convert colourless compound p-nitro phenol phosphate (PNPP) into yellow colour compound p- nitro-phenol (PNP) and also offers a means of signal amplification.

Thus, rather than the detection system relying on autoradiography, which is necessary for radiolabels, a series of reaction resulting in colour, a light or a chemiluminescence are much better, since autoradiography may take 1-3 days, where colour and chemiluminescence reaction takes a few minutes.

End Labelling of DNA Molecules:

The simplest form of labelling is by 5′- or 3′-end labelling. 5′-end labelling involves a phosphate transfer or exchange reaction where the 5′-phosphate of the DNA to be used is replaced by 32 P. This is carried by two enzymes: the first, alkaline phosphatase removing phosphate group followed by poly nucleotide kinase catalyzing the transfer of phosphate group ( 32 P) to 5′ end of DNA. The newly labelled probe is then purified, usually by chromatography through a sephadex column to remove any unincorporated radiolabel.

Using other end of DNA molecule, the 3′- end is slightly less complex. Here a new, labelled dNTP ([a 32 -P]ATP or biotin-labelled dNTP) is added to the 3′-end of DNA by enzyme termi­nal transferase. Although this is a simpler reac­tion, a potential problem exists because a new nucleotide is added to the existing sequence and so the complete sequence of the DNA is altered, which may affect its hybridization to its target sequence. End-labelling methods also suffer from the fact that only one label is added to DNA so such methods are of low activity in comparison to other who incorporate label throughout the length of sequence of DNA.

Random Primer Labelling:

The DNA to be labelled is first denatured and is then allowed to re-natured in presence of random sequences of hexamers or hexanucleotide. These hexamers will by chance bind to DNA sample wherever they encounter a complementary sequence and so the DNA will acquire hexanucleotide annealing to it.

Each of the hexamers can act as primer for synthesis of a fresh strand of DNA catalysed by DNA polymerase since it has free 3′- hydroxyl group. The Klenow fragment of DNA polymerase is used for random primer labelling since it lacks 5′ → 3′ exonuclease activity but still acts as 5′ → 3′ polymer­ase.

Nick Translation:

It is a traditional method of labelling DNA. Low concentrations of DNase I is used to create occasional single strand nick which is then filled in by DNA polymerase using an appropri­ate dNTP at the same time making a new nick at 3′-side of the previous one. If labelled dNTPs are used, the DNA can be labelled to a very high specific activity.

2: Chemistry and Biologically Important Molecules

A. Chemical bonds/attractive forces: attractive forces between atoms. Number of electrons in outermost energy level /shell determines chemical properties of an atom. If outer (=valence) shell is full of electrons, atom does not tend to react with other atoms. If valence shell is not full, atom tends to react with other atoms to fill valence electron shell.

Three types of bonds/attractive forces: Type of bond formed depends on electron configuration and electronegativity of atoms involved:

1. ionic bonds: sodium and chloride ions. Sodium atom (11 p+, 11e-) loses 1 electron and becomes positively charged cation. Chloride atom (17 p+, 17 e-) gains 1 electron and becomes negatively charged anion.

Oppositely charged sodium and chloride ions attracted to each other. Electrical attraction between 2 oppositely charged ions is an ionic bond.

2. covalent bonds: formed when atoms share pairs of electrons to fill valence shell. Number of covalent bonds formed depends on valence electrons. single, double, triple covalent bonds. e.g. H2O. Indicated by solid line &ldquo-&ldquo

a. electronegativity: - atoms ability to attract electrons. low= H, C high=O

b. polar covalent bond: 2 atoms unequally share electrons one member carries slight positive charge, one member carries slight negative charge. eg water

partial charge=&delta

c. nonpolar covalent bond: 2 atoms equally share electrons. Charge evenly distributed. eg hydrocarbons

If difference of electronegativity is approx >0.5, covalent bond formed will be polar

3. Hydrogen "bonds" (&ldquo. &rdquo): Not true bonds, an attractive force. Hydrogen involved in a polar covalent bond carries a partial &delta positive charge. The hydrogen may also be attracted to other molecules carrying a partial &delta negative charge, forming hydrogen bond.

Bio 440 Chemical Bonds/attractive forces concept map : distribute in lecture

B. Bond strength in aqueous solutions: strongest covalent> ionic> H bond weakest

1. Although weak, multiple hydrogen bonds are important in stabilizing the three-dimensional shape of many biological molecules. (Shape determines function &ldquofunctional conformation&rdquo)

2. Covalent bonds are usually very stable

-cells use protein catalysts called enzymes to &ldquobreak&rdquo covalent bonds e.g. hydrolysi

3. Ionic compounds &ldquofall apart&rdquo in water. ex NaCl crystals in water ions attracted to polar water molecules

IV. Special Properties of Water

Most microorganisms contain approx. 70% water. Most chemical reactions occur in aqueous environments

Key to unique properties of water: polarity of water molecule and hydrogen bond formation with other water molecules

2. less dense as solid (ice) than liquid therefore ice floats-> oceans/lakes habitable in cold climates

3. high specific heat/water absorbs lots of energy before enough H bonds are broken t increase kinetic energy/velocity of water molecules leading to increase temperature. Water helps moderate temperature changes.

4. high heat of vaporization/as water evaporates, removes energy/heat-> cooling effect

5. excellent solvent=polar molecule

hydrophilic substances=water &ldquoloving&rdquo, polar/ionic substances water molecules attracted to, form bonds with hydrophilic substances

hydrophobic substances=water &ldquohating&rdquo noncharged, nonpolar, do not dissolve in /mix with water since these substances lack charges, water molecules are not attracted to them e.g hydrocarbons, lipids

6. water ionizes: H2O <-> H + + OH -

7. Water and acid-base balance. pH (also Ch 4 in lab manual)

pH= -log [H + ] (more in lab)

acids : fig ___-acids ionize, release free H + , increase H + concentration of aqueous solution

bases : lower H + concentration of aqueous solution (strong bases ionize releasing hydroxyl ions OH- e.g. NaOH-> Na + + OH - weak bases bind free hydrogen ions/protons H + e.g. ammonia NH3 + H+-> NH4 +

V. Functional Groups and Carbon skeletons

A. Organic chemistry: study of carbon containing compounds (excluding inorganic CO2)

1. carbon/carbon skeletons: ability to form 4 covalent bonds/endless variety of carbon skeletons

-variations of carbon number, branching, double bonds, ring formation

-examples hydrocarbons, = H and C only nonpolar: methane, ethane

2. adding functional groups to carbon skeletons changes character of molecule most often chemical reactions involve functional groups

B. Functional Groups: (R=&rdquorest&rdquo of molecule/carbon skeleton)

methyl: nonpolar, hydrophobic R-CH3

hydroxyl: polar, hydrophilic R-OH

carbonyl C=O : polar, hydrophilic

terminal carbonyl: aldehydes: R-CH=O

internal carbonyl ketone: R1-CO-R2

carboxyl: polar, hydrophilic, acidic/ionizes to increases free H + in solution

amino: polar, hydrophilic, basic, binds H + , decreases free H + in solution

phosphoric acid residue (very acidic)->ionizes->phosphate: polar, hydrophilic,

VI. Biologically Important Molecules: proteins, nucleic acids, carbohydrates, lipids

(see chem. homework sheet )

A. Macromolecules= &ldquolarge molecules&rdquo

1. most are polymers of repeating subunits (monomers)

-joined by removal of H + OH = water : &ldquodehydration synthesis&rdquo or &ldquocondensation reactions&rdquo (opposite=hydrolysis)

A-H + OH-B--> -dehydration synthesis--> A-B + H2O

a. proteins/amino acids

b. nucleic acids/nucleotides

c. carbohydrates: polysaccharides/monosaccharides &ldquosugars&rdquo

3. Lipids=hydrophobic

B. Proteins: polymers of amino acids

1. Amino acids:

20 different types/all share basic structure:

-central carbon with 4 &ldquogroups&rdquo attached H, carboxyl group, amino group and R side group.

20 different R side groups

-R groups specify &ldquopersonality&rdquo/chemical reactivity of amino acid.

-Amphoteric: has both acidic and basic qualities

-Proteins contain L isomers only

.( Bacterial cell walls/some antibiotics contain D amino acids these are NOT proteins).

-Bacteria have formyl-methionine f-met as 21 st amino acid

2. Polymers of amino acids: aa1-aa2-aa3-aa4-aa5-aa6---- linked by peptide bonds

3. most abundant component of cells (next to water) 50% of dry weight=protein

4. Functions:

a. enzymes= protein catalysts/speed up chemical reactions without being &ldquoused up&rdquo -biochemical &ldquotools&rdquo of cells active site=substrate specific

c. transport proteins d. receptors, antibodies, chemical messengers and more.

5. Protein structure: primary, secondary, tertiary, quaternary.

Levels of protein structure

Primary structure : specific sequence of amino acids in polypeptide chain

-determines all other higher levels of structure thus determines &ldquofunctional conformation&rdquo

-amino acid sequence of a protein is determined by the DNA nucleotide base sequence of its gene

Secondary structure coiling/folding of polypeptide chain into specific patterns such as alpha &alpha helix /&beta pleated sheets

-hydrogen bonds between members of peptide &ldquobackbone&rdquo (R groups not involved)

Tertiary structure : additional folding of polypeptide into complex 3-D shape- for many proteins, the functional conformation. R group interactions (H bonds, ionic, covalent bonds, hydrophobic interactions, London Dispersion Forces/van der Waals forces disulfide bridges between cysteine residues)

Quaternary structure : the association of 2 or more polypeptide subunits to form functional protein. ex hemoglobin, immunoglobulin. R group interactions as for tertiary structure

6. Protein Denaturation: unfolding of polypeptides. Loss of shape/structure=loss of function

-high temperature, pH extremes, heavy metals, alcohols.

-applications: protein denaturation is used to inactivate/kill pathogenic microbes.

- autoclave, boiling, alcohols, heavy metals, physical "abuse"

-Usually denaturation is irreversible.

-Exceptions: thermophiles, chaperone proteins.

7. &ldquoProtein mis-folding diseases&rdquo: abnormally folded prion proteins cause TSE, Transmissible Spongiform Encephalopathies. The disease-causing prions are incredibly difficult to denature, resist denaturation by cooking, normal autoclaving, most chemicals. Most resistant biological structures known. Additional protein mis-folding disease: Alzheimer's, Parkinsons, more (could Alzhemimer's, Parkinson's diseases be transmissible as are TSE's? Could the misfolded proteins be resistant to denaturation, thus could be transferred by medical instruments, dental instruments. ).

8. Antibiotics inhibiting bacterial protein synthesis: . examples include tetracycline, aminoglycosides, chloramphenicol, macrolides ( eg erythromycin) lincosamide, streptogramins

9. Antiviral drugs: some drugs used to inhibit replication of viruses interfere with protein synthesis/processing eg antisense nucleic acids, protease inhibitors

C. Nucleic Acids

1. Polymers of nucleotides

a. DNA= deoxyribonucleic acid genetic information of cell

b. RNA= ribonucleic acid: messenger, (mRNA), ribosomal (rRNA) and transfer (tRNA). Functions in transcription and translation of DNA base sequences into amino acid sequences of proteins

2. Nucleotides: 3 components

a. 5 C sugar + phosphate group + nitrogenous base (purine or pyrimidine)

b. complementary base pairing A::T A::U C::G

c. create a table to compare and contrast DNA to RNA

nitrogenous bases adenine A adenine A

Complementary base pairing

#strands double stranded=ds single stranded=ss

3. DNA , RNA: polymers of nucleotides joined by phosphodiester bonds. Hydrogen bonds between complementary bases hold 2 complementary sister DNA strands together (intermolecular H bonds) and stabilize complex 3-D shapes of RNA (intramolecular H bonds)

4. a. Cells carry both DNA and RNA. In contrast, viruses carry either DNA or RNA, not both (some exceptions). There are some ssDNA viruses (parvovirus) and some ds RNA viruses (reoviruses) as well as ds DNA viruses and ssRNA viruses

b. Most prokaryotes carry chromosomes of circular ds DNA most eukaryotes carry multiple linear chromosomes made of ds DNA

c. DNA can form complementary H bonds with RNA. This process is crucial in transcription (more later)

d. To distinguish carbons in the 5 carbon sugar from the nitrogenous base carbons in a nucleotide, a &ldquo&rsquo&rdquo is added to the sugar carbons to distinguish them from the nitrogenous base carbons. Thus carbon 3&rsquo or 5&rsquo refers to carbons in the sugar and carbon 3 or carbon 5 refers to carbons in the nitrogenous base

e. The combination of a 5 carbon sugar and a nitrogenous base is called a nucleoside. 1,2, or 3 phosphate groups made be added to the 5&rsquo carbon creating nucleoside-mon-, di- or tri-phosphates

f. Phosphates groups are attached to the 5&rsquo carbon of the sugar in a nucleotide. In a growing strand of nucleic acid, incoming nucleotides may only be added at the 3&rsquo OH end of the strand. Thus a strand is said to elongate in a 5&rsquo to 3&rsquo direction (5&rdquo->3&rsquo)

g. If cut, a strand of DNA or RNA has a free 5&rsquo phosphate end (5&rsquoP) and a free 3&rsquoOH end. In ds DNA, complementary &ldquosister&rdquo strands are &ldquoantiparallel&rdquo in that the 5&rsquo P end of one strand lies adjacent to the 3&rsquoOH end of the complementary sister strand

h. Energized precursors required for synthesis of DNA/RNA are nucleoside triphosphates in RNA/DNA strands, nucleoside monophosphate residues are present (more later)

i. some antifungal drugs interfere with RNA function e.g. flurocytosine. Some antiprotozoan drugs inhibit nucleic acid synthesis/function: eflornithine, nitroimadazoles, pentamidine. Some antihelminthic drugs inhibit nucleic acid synthesis: niridazole, olitpraz, oxamniquine, all for Schistosoma infections

i. Nucleoside triphosphates as energy sources: ATP: ATP is the &ldquoprinciple short-term, recyclable energy source for cells. Energy is stored in high energy bonds between phosphate groups 1

tilde indicates bind which releases large amounts of energy when hydrolyzed . More later in metabolism. Adenosine triphosphates is also a building block for RNA

D. Carbohydrates : carbon + water/ generalized formula (CH2O)n. Energy storage, structural. C skeleton, carbonyl group, hydroxyl groups. Suffix &ldquo-ose&rdquo (see chem. homework sheet)

a. monosaccharides (=monomers) ex glucose, fructose, galactose, ribose, deoxyribose- structure of glucose & fructose . In aqueous solutions, glucose alternates between linear and ring forms. 2 ring forms depending on orientation of &ndashOH on carbon 1, alpha and beta.

-glucose is used as a source of energy and carbon

b. disaccharides: ex sucrose (glucose + fructose), lactose (glucose + galactose)

-glycosidic bonds join monosaccharide residues

c. polysaccharides: polymers of monosaccharides covalent bonds=glycosidic bonds. e.g. glycogen, starch (amylose + amylopectin)=polymers of alpha glucose helical,some branched, cellulose = polymers of beta glucose straight chains (know)

2. Modified sugars e.g. modified glucose

-NAG: addition of amino residue and acetyl group to carbon 2 of glucose forms

N-acetylglucosamine (NAG)

-chitin: polymer of beta-NAG found in cell walls of fungi and exoskeleton of arthropods. Function similar to cellulose in plant cell walls

-NAM : further modification of NAG to form N-acetylmuramic acid, NAM (lactic acid residue covalently linked to carbon3).. Only members of Domain Bacteria have enzymes to synthesize NAM, thus NAM is called a &ldquosignature&rdquo molecule&rdquo for Doman Bacteria

-peptidoglycan: Unique to members of Domain Bacteria, found in bacterial cell walls, functions to strengthen cell walls and prevent osmotic lysis (similar to chitin and cellulose). Polymers of alternating NAG and NAM (&ldquoglycan&rdquo sugars) linked by beta glycosidic bonds crosslinked by peptide chains (&ldquopeptido&rdquo). Peptidoglycan/pg synthesis is the target of many antibiotics e.g. beta lactam antibiotics penicillin, ampicillin etc and vancomycin, cycloserine, bacitracin

3. Hydrolysis of carbohydrates: an organism&rsquos ability to use a carbohydrate as a carbon and energy source depends on its ability to synthesize hydrolytic enzymes specific for a carbohydrate

Mammalian &ldquolactase&rdquo/bacterial &ldquobeta-galactosidase&rdquo

Maltose (breakdown product of starch)

4. Hydrolysis of glucose polymers by mammals: Mammals have enzymes to hydrolyze alpha glycosidic bonds between alpha glucose subunits found in starch and glycogen (e.g. salivary amylase). However mammals lack cellulase to hydrolyze beta glycosidic bonds found in cellulose, consequently mammals cannot digest plant cellulose (&ldquofiber&rdquo).

-Some herbivores (animals who eat plants) can digest cellulose-how?

One group, the ruminants, have a 4 compartment &ldquostomach&rdquo or digestive chamber which includes a rumen and reticulum. The rumen-reticulum are microbial fermentation vats filled with bacteria and protozoa. These microbes synthesize cellulase and hydrolyze cellulose the ruminants eat, releasing glucose subunits which can then be fermented. As a result of glucose fermentation, the microbes produce large amounts of carbon dioxide and methane (&ldquogreenhouse gases&rdquo released by &ldquoeructation&rdquo) and short chain fatty acids (C/energy source) and ammonium (absorbed across rumen wall into bloodstream). Rumen fluid then flows into the true stomach, the abomasum, for further digestion (note: the microbes themselves can act as nutrients). Thus the symbiotic relationship between ruminant and rumen microbe is mutualistic, both partners benefit.

-Although horses and rabbits are not ruminants, their large ceca -(singular=cecum) contain cellulose digesting microbes and serve a similar function as the rumen-reticulum.

-Of interest, rabbits are coprophagic, they eat their feces, thus benefiting from digestion of the cecal microbes passed in the feces.

-recombinant cellulase made by E.coli:

E. Lipids: primarily nonpolar, hydrophobic molecules

1. Functions: important part of cell membranes, energy reserves, messenger molecules

2. Two categories: fatty acid containing and fatty acid lacking

a. fatty acid containing lipids

i.simple lipids= fats (aka triglycerides, triacylglycerol) fats=solid at room temperature oils= liquids at room temp. Energy reserves.

- fats/oils= glycerol + 3 fatty acids

- fatty acid= carboxyl group + hydrocarbon tail (nonpolar, hydrophobic tail)

-: fatty acids vary in #C&rsquos in tail and presence of C=C double bonds

(unsaturated fatty acids) or lack of C=C bonds (saturated fatty acids)

ii. Complex lipids=phospholipids: glycerol + 2 fatty acids + phosphate + &lsquoR&rdquo

- polar, hydrophilic &ldquohead&rdquo + nonpolar, hydrophobic &ldquotail&rdquo

- amphipathic/amphiphilic: contains both hydrophilic and hydrophobic regions

- in aqueous solutions, phospholipids can spontaneously form phospholipid bilayers

-important component of cell membranes

iii. waxes fatty acids + alcohols. Acid-fast bacteria &lsquoAFB&rsquos&rdquo such as Mycobacterium tuberculosisand Mycobacterium leprae (Hansen&rsquos Disease/leprosy ) synthesize &ldquowaxy&rdquo cell walls (arabinogalactan-mycolic acid) which protect them against drying and chemicals such as disinfectants and antibiotics. As the hydrophobic waxy layer inhibits passage of antibiotics, people treated for TB/leprosy must take antibiotics for many months/years. AFB grow slowly in the lab and are hard to stain (require special &ldquoacid-fast&rdquo stain -more in lab). Isoniazid and ethambutol inhibit formation of the waxy cell wall layer.

b. fatty acid -lacking lipids

i. steroids=lipids composed of 4 HC rings + functional groups

-. e.g. cholesterol component of animal/protozoa cell membranes, precursor of hormones in animals (estrogen, testosterone, vitamin D

-***not found in most bacteria (***exceptions ex Mycoplasma)

- e.g. fungal sterols of cell membrane ex ergosterol.

-target of polyene antifungal agents ex Nystatin. Amphotericin B

-azole antifungal agents such as miconazole, fluconazole target fungal sterol synthesis

Create summary charts (partially completed example below) . Be able to identify molecules: amino aids, polypeptide, glucose, a glucose polymer, DNA, RNA, fat/oil, phospholipid, sterol. The only molecules you will be asked to draw from memory will be reaction between 2 amino acids forming dipeptide. Do chem. homework sheet posted on D2L

Why are nucleotides added to 3' end?

The DNA is only copied in the 5' to 3' direction because eukaryotic chromosomes have many origins for each chromosome in keeping with their much larger size. If some were copied in the other direction, mistakes will happen. It keeps every cell division on the same page, so to speak.

Because DNA synthesis can only occur in the 5' to 3' direction, a second DNA polymerase molecule is used to bind to the other template strand as the double helix opens. This molecule synthesizes discontinuous segments of polynucleotides, called Okazaki fragments. Another enzyme, called DNA ligase, is responsible for stitching these fragments together into what is called the lagging strand.

The mechanism of DNA ligase is to form two covalent phosphodiester bonds between 3' hydroxyl ends of one nucleotide, ("acceptor") with the 5' phosphate end of another ("donor"). The two "sticky ends" have to be in opposite directions for replication of the entire DNA molecule to be complete.

The average human chromosome contains an enormous number of nucleotide pairs that are copied at about 50 base pairs per second. Yet, the entire replication process takes only about an hour. This is because there are many replication origin sites on a eukaryotic chromosome. Therefore, replication can begin at some origins earlier than at others. As replication nears completion, "bubbles" of newly replicated DNA meet and fuse, forming two new molecules.

Explain what 5' and 3' ends mean in regards to DNA structure?

5' and 3' ends describes the directionality of the DNA molecule. Essentially, the strand of a DNA molecule can have a 5' end and a 3' end. To understand what a 5' or 3' end is, we need to look at the molecular structure of DNA. DNA is a polymer of nucleotides, where each nucleotide is made up of a sugar (deoxyribose), a nitrogenous base, and a phosphate group. The deoxyribose sugar is a 5 carbon structure, where each carbon can be numbered 1-5. The base is always connected to Carbon 1 of the sugar and the phosphate group is connected to Carbon 5 of the sugar. The nucleotides are then connected to one another to form the polymer whereby the phosphate group of one nucleotide (on Carbon 5) connects to the next nucleotide sugar via Carbon 3. Therefore when the strand is built, the top nucleotide will have a free phosphate group on the Carbon 5 of the sugar, hence the 5' end, and the last nucleotide of the strand will have a free OH on the Carbon 3 of the sugar. Since DNA is made up of two antiparallel strands, each strand can have its own directionality. As they are antiparallel, they will run in opposite directions.


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