bio.lesson document 7

8/6/2019 bio.lesson document 7

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BIO 203 Biochemistry Iby

Seyhun YURDUGL,Ph.D.

Lecture 8Nucleic Acids


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C

ontent outline Nucleosides and nucleotides

Synthetic nucleotide analogues Polynucleotides

Thermal properties of DNA

Electrophoresis


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Introduction

Nucleotides: one of the most importantmetabolites of the cell.

found primarily as the monomeric unitscomprising the major nucleic acids of the cell,RNA and DNA.

also are required for numerous other importantfunctions within the cell.


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These functions include: 1. serving as energy stores for future use in

phosphate transfer reactions.

These reactions are predominantly carriedout by ATP.

2. forming a portion of several important

coenzymes: such as NAD+, NADP+, FAD and coenzymeA.


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These functions include: 3. serving as mediators of numerous important

cellular processes:

such as second messengers in signal transductionevents. The predominant second messenger: Cyclic AMP (cAMP), a cyclic derivative of AMP

formed from ATP. 4. controlling numerous enzymatic reactions

through allosteric effects on enzyme activity.


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These functions include: 5. serving as activated intermediates in

numerous biosynthetic reactions:

include S-adenosylmethionine (S-AdoMet)involved in methyl transfer reactions,

as well as the many sugar coupled

nucleotides involved in: glycogen and glycoprotein synthesis.


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Nucleoside and nucleotide

structure and nomenclature:

derivatives of the heterocyclic highly basic,compounds:

purine and pyrimidine.

PurinePyrimidine


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Structure It is the chemical basicity of the nucleotides

that has given them the common term

"bases" as they are associated with nucleotides

present in DNA and RNA.


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Structure In cells: Five major bases.

The derivatives of purine are called adenineand guanine,

and the derivatives of pyrimidine are calledthymine, cytosine and uracil.

The common abbreviations used for thesefive bases are, A, G, T, C and U.


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Base Formulas (Basesubstitution:X=H)

Cytosine, C

Uracil, U

Thymine, T

Adenine, A

Guanine, G


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Nucleoside forms If X=ribose or deoxyribose

Cytidine

Uridine

Thymidine

Adenosine Guanosine


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Nucleotide forms If X=ribose phosphate

Cytidine monophosphate (CMP)

Uridine monophosphate (UMP)

Thymidine monophosphate (TMP)

Adenosine monophosphate (AMP) Guanosine monophosphate (GMP)


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Nucleosides The purine and pyrimidine bases in cells:

linked to carbohydrate and in this form:

termed as nucleosides. The nucleosides are coupled to D-ribose or 2'-

deoxy-D-ribose;

through a -N-glycosidic bond:

between the anomeric carbon of the ribose and the N9 of a purine or N1 of a pyrimidine.


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Nucleotides The base:

can exist in 2 distinct orientations about the N-

glycosidic bond. These conformations :

identified as,syn

and anti.

Anti conformation predominates in naturallyoccurring nucleotides.


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Syn and anti conformations

syn-Adenosine anti-Adenosine


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Nucleotides found in the cell primarily in their

phosphorylated form.

The most common site of phosphorylation ofnucleotides found in cells:

the hydroxyl group attached to the 5'-carbon ofthe ribose.


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Nucleotides The carbon atoms of the ribose present in

nucleotides:

designated with a prime (') mark to distinguishthem from the backbone numbering in thebases.

Nucleotides can exist in the mono-, di-, or tri-phosphorylated forms.


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Nucleotides given distinct abbreviations to allow easy

identification of their structure,

and state of phosphorylation.

The monophosphorylated form of adenosine(adenosine-5'-monophosphate):

written as, AMP.


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Nucleotides The di- and tri-phosphorylated forms:

written as, ADP and ATP, respectively.

The use of these abbreviations assumes that:

the nucleotide is in the 5'-phosphorylatedform.


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Nucleotides The di- and tri-phosphates of nucleotides:

linked by acid anhydride bonds.

Acid anhydride bonds: have a high potential to transfer the

phosphates to other molecules.

due to this property: the nucleotides thatresults in their involvement in grouptransfer reactions in the cell.


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Nucleotides The nucleotides found in DNA:

unique from those of RNA in that;

the ribose exists in the 2'-deoxy form and theabbreviations of the nucleotides:

contain a d- designation.

The monophosphorylated form of adenosinefound in DNA (deoxyadenosine-5'-monophosphate) is written as dAMP.


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Nucleotides The nucleotide uridine:

never found in DNA and thymine is almostexclusively found in DNA.

Thymine: found in tRNAs but not rRNAsnor mRNAs.

There are several less common bases foundin DNA and RNA.


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Nucleotides The primary modified base in DNA is 5-

methylcytosine.

A variety of modified bases appear in thetRNAs.

Many modified nucleotides:

encountered outside of the context of DNAand RNA;

that serve important biological functions.


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Adenosine derivatives

The most common adenosine derivative is the cyclicform,

3'-5'-cyclic adenosine monophosphate, cAMP. This compound is a very powerful second

messenger,

involved in passing signal transduction events fromthe cell surface to internal proteins,

e.g. cAMP-dependent protein kinase, PKA


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Adenosine derivatives

The most important cGMP coupled signal transductioncascade:

photoreception.

in this case activation of rhodopsin (in the rods); or other opsins (in the cones);

by the absorption of a photon of light;

through 11-cis-retinal covalently associated with rhodopsinand opsins

activates transducin,

which in turn activates a cGMP specific phosphodiesterase:that hydrolyzes cGMP to GMP.


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Synthetic nucleotide analogues

Many nucleotide analogues : chemically synthesized and used for their therapeutic

potential.

nucleotide analogues : can be utilized to inhibit specificenzymatic activities.


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A large family of analogues are used as anti-tumor agents:

because they interfere with the synthesis ofDNA

and thereby preferentially kill rapidly dividingcells such as tumor cells.


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Some of the nucleotide analogues commonlyused in chemotherapy:

6-mercaptopurine, 5-fluorouracil,

5-iodo-2'-deoxyuridine

6-thioguanine.


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Each of these compounds :

disrupts the normal replication process,

by interfering with the formation of correctWatson-Crick base-pairing.


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Nucleotide analogues also have been targeted foruse as antiviral agents.

Several analogs are used to interfere with thereplication of HIV:

such as AZT (azidothymidine),

and ddI (dideoxyinosine).


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Several purine analogs: used to treat gout;

e.g. most common : allopurinol;

which resembles hypoxanthine. Allopurinol inhibits the activity of xanthine oxidase,

an enzyme involved in de novopurine biosynthesis.


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Additionally, several nucleotide analoguesare used:

after organ transplantation

in order to suppress the immune system,

and reduce the likelihood of transplantrejection by the host.


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Polynucleotides

formed by the condensation of two or morenucleotides.

condensation most commonly occursbetween the alcohol of a 5'-phosphate ofone nucleotide;

and the 3'-hydroxyl of a second, with the elimination of H2O, forming a

phosphodiester bond.


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Polynucleotides The formation of phosphodiester bonds in

DNA and RNA:

exhibits directionality.

The primary structure of DNA and RNA(the linear arrangement of the nucleotides):

proceeds in the 5' ----> 3' direction.


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Polynucleotides The common representation of the primary

structure of DNA or RNA:

write the nucleotide sequences from left toright synonymous with the 5' -----> 3'direction as shown:

e.g. 5'-pGpApTpC-3'


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Structure of DNA

Utilizing X-ray diffraction data, obtained fromcrystals of DNA,

James Watson and FrancisC

rick proposed a modelfor the structure of DNA.


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Structure of DNA

This model predicted that DNA would exist:

as a helix of two complementary antiparallel strands,

wound around each other in a rightward direction, and stabilized by H-bonding between bases in

adjacent strands.


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S

tructure of DNA

In the Watson-Crick model,

the bases are in the interior of the helix

aligned at a nearly 90 degree angle relativeto the axis of the helix.

Purine bases form hydrogen bonds withpyrimidines,

in the crucial phenomenon of base pairing.


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Structure of DNA

Experimental determination has shown that,in any given molecule of DNA,

the concentration of adenine (A) is equal tothymine (T)


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Structure of DNA

and the concentration of cytidine (C) isequal to guanine (G).

This means that A will only base-pair withT;

and C with G.


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Structure of DNA

According to this pattern, known as Watson-Crick base-pairing:

the base-pairs composed of G and C containthree H-bonds,

whereas those of A and T contain two H-bonds.

This makes G-C base pairs more stable thanA-T base-pairs.


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A-T Base Pair

G-C Base Pair


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The antiparallel nature of the helix:

stems from the orientation of the individual

strands. From any fixed position in the helix,

one strand is oriented in the 5' ---> 3'

direction and the other in the 3' ---> 5' direction.

Structure of DNA


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Structure of DNA

On its exterior surface,

the double helix of DNA contains two deep

grooves:

between the ribose-phosphate chains.

These two grooves are of unequal size;

and termed the major and minor grooves.


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Structure of DNA

The difference in their size is:

due to the asymmetry of the deoxyribose

rings; and the structurally distinct nature of the

upper surface of a base-pair;

relative to the bottom surface.


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Structure of DNA

The double helix of DNA (A-form)

has been shown to exist in several different

forms, depending upon sequence content and ionic

conditions of crystal preparation.


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Structure of DNA

The B-form of DNA:

prevails under physiological conditions of

low ionic strength; and a high degree of hydration.


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Structure of DNA

Regions of the helix that are rich in pCpGdinucleotides:

can exist in a novel left-handed helicalconformation termed Z-DNA.

This conformation results from a 180 degreechange in the orientation of the bases,

relative to that of the more common A- and B-DNA.


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B-DNA Z-DNA


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As cells divide, it is a necessity that the DNA be copied

(replicated), in such a way that each daughter cell: acquires the same amount of genetic material.


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In order for this process to proceed:

the two strands of the helix must first be

separated, in a process termed denaturation.

This process can also be carried out in vitro.


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If a solution of DNA :

subjected to high temperature, the H-bonds

between bases become unstable; and the strands of the helix separate in a

process of thermal denaturation.


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The base composition of DNA:

varies widely from molecule to molecule

and even within different regions of thesame molecule.

Regions of the duplex that have

predominantly A-T base-pairs: will be less thermally stable than;

those rich in G-C base pairs.


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In the process of thermal denaturation,

a point is reached at which 50% of the DNA

molecule exists as single strands. This point is the melting temperature (TM),

and characteristic of the base composition

of that DNA molecule.


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The TM depends upon several factors inaddition to the base composition.

These include the chemical nature of thesolvent;

and the identities and concentrations of ions inthe solution.


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When thermally melted DNA: cooled,

the complementary strands will again re-form the

correct base pairs, in a process termed annealing or hybridization.

The rate of annealing:

dependent upon the nucleotide sequence of thetwo strands of DNA.


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Analysis of DNA structure

Chromatography:

Several of the chromatographic techniquesavailable for the characterization of

proteins; can also be applied to the characterization

of DNA.

The most commonly used technique: HPLC (high performance liquidchromatography).


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Analysis of DNA structure

Affinity chromatography technique can be alsoemployed.

One common affinity matrix: hydroxyapatite (aform of calcium phosphate),

which binds double-stranded DNA;

with a higher affinity than single-stranded DNA.


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Electrophoresis:

This procedure:

serve the same function with regard to DNA

molecules; as it does for the analysis of proteins.

However, since DNA molecules have much

higher molecular weights than proteins, the molecular sieve used in electrophoresisof DNA must be different as well.


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Electrophoresis:

The material of choice for DNA:

agarose, a carbohydrate polymer purified

from a salt water algae. is a copolymer of mannose and galactose

that when melted and re-cooled:

forms a gel with pore sizes; dependent upon the concentration ofagarose.


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Electrophoresis:

The phosphate backbone of DNA:

highly negatively charged,

therefore DNA will migrate in an electricfield.

The size of DNA fragments:

can then be determined by comparing theirmigration in the gel to known sizestandards.


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Pulsed-field gel electrophoresis

Extremely large molecules of DNA (inexcess of 106 base pairs):

effectively separated in agarose gels usingpulsed-field gel electrophoresis (PFGE).


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Pulsed-field gel electrophoresis

This technique employs two or more electrodes,

placed orthogonally with respect to the gel,

that receive short alternating pulses of current. PFGE allows whole chromosomes:

and large portions of chromosomes to be analyzed.


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LITERATURE CITED

Devlin,T.M. Textbook of Biochemistry withClinical Correlations,Fifth Edition,Wiley-LissPublications,New York, USA, 2002.

Lehninger, A. Principles of Biochemistry, Secondedition, Worth Publishers Co., New York, USA,1993.

Matthews, C.K. and van Holde, K.E.,Biochemistry, Second edition, Benjamin /Cummings Publishing Company Inc., SanFrancisco, 1996.

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