DNA structure- Part I

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Nucleotides and Nucleic AcidsBiological function of nucleotides and nucleic acidsStructures of common nucleotidesStructure of double-stranded DNAStructures of ribonucleic acidsDenaturation and annealing of DNAChemistry of nucleic acids; mutagenesisKey topics :

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Road to the Double Helix Franklin and Wilkins Cross means helixDiamonds mean that the phosphate- sugar backbone is outside Calculated helical parameters

Watson and Crick Missing layer means alternating pattern (major & minor groove) Hydrogen bonding: A pairs with T G pairs with CDouble helix fits the data!

Watson, Crick, and Wilkins shared 1962 Nobel PrizeFranklin died in 1958

X-ray crystallography of DNAM. Wilkins , R. Franklin, R. Gosling

X-crystallograpy of A, B form of DNA results in X-shape diffraction pattern, indicating that DNA molecules are helical with two periodicities along the long axes, a primary one of 3.4 and a secondary one of 34.*2-*

Discovery of DNA

In 1953, Watson and Crick, used x-ray diffraction data obtained by Franklin to deduce DNAs structure.

It was immediately apparent to scientists how this unique structure of DNA allow biological information to be easily and faithfully duplicated and transmitted from generation to another.

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Discovery of RNA Similar to DNA, RNA was first isolated in nineteenth century from the nuclei of cells. Scientists later recognized that RNA is chemically distinct from DNA. *2-*

Continue

Neighboring -OH groups (2' and 3') in RNA make it more susceptible to hydrolysis. DNA, lacking 2'-OH is more stable. This makes sense - the genetic material must be more stable.RNA is designed to be used and then broken down.

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So. The structural stability of DNA and the structural diversity of RNAs explain why these molecules have evolved to function in all aspects of maintaining and transmitting biological information.*2-*

Functions ofNucleotides and Nucleic AcidsNucleotide Functions:Energy for metabolism (ATP)Enzyme cofactors (NAD+)Signal transduction (cAMP)

Nucleic Acid Functions: Storage of genetic info (DNA)Transmission of genetic info (mRNA)Processing of genetic information (ribozymes)Protein synthesis (tRNA and rRNA)

Nucleotides and NucleosidesNucleotide = Nitrogeneous basePentosePhosphate

Nucleoside = Nitrogeneous basePentose

Nucleobase = Nitrogeneous base

The properties of NucleotidesA nucleotide is molecule consisting of three characteristic components:I. a heterocyclic base.II. a five- carbon sugar (pentose).III. a phosphate group.

The same molecules without the phosphate group is called a nucleoside.

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Phosphate GroupNegatively charged at neutral pH Typically attached to 5 positionNucleic acids are built using 5-triphosphatesATP, GTP, TTP, CTPNucleic acids contain one phosphate moiety per nucleotideMay be attached to other positions

I. Pyrimidine/Purine Bases Each base is a derivative of one of two parent compounds, a Purine or a Pyrimidine, which are nitrogenous bases. They are called bases because free purines and pyrimidines are weakly basic compounds.

The carbon and nitrogen atoms are numbered according to convention to facilitate the naming and identification of the any other derivative compounds.

Pyrimidine Bases Cytosine is found in both DNA and RNA Thymine is found only in DNA Uracil is found only in RNA All are good H-bond donors and acceptors Cytosine pKa at N3 is 4.5 Thymine pKa at N3 is 9.5 Neutral molecules at pH 7

Purine BasesAdenine and guanine are found in both RNA and DNAAlso good H-bond donors and acceptors Adenine pKa at N1 is 3.8Guanine pKa at N7 is 2.4Neutral molecules at pH 7

Minor Bases Less common bases can occur. Principally but not exclusively, in transfer RNAs.*2-*

Base and nucleotides derivativesStimulant: caffeine-trimethylated purine inhibits natural ligands for adenosine A2 receptor which regulates sleepiness & voluntary movement.Anti-cancer drug: 6-mercaptopurine(6-MP) & 5-fluorouracil (5-FU), blocks the synthesis of nucleic acids. Anti-viral drug: Acyclovir, inhibit synthesis of nucleic acids, treatment of herpes virus. Anti-AIDS drug: lacks 2 OH and inhibit virus DNA replication.

keto-enol tautomerism of uraciltautomerization of guanine*2-*Prototropic tautomers are structural isomers that differ in the location of protonsKeto-enol tautomerism is common in ketonesBoth tautomers exist in solution but the keto forms are predominant at neutral pHTautomerism of Nucleobases

Numbering system of nucleic acids.

a. phosphate (alpha, beta, gamma)b. base (1,2,3,...)c. sugar (1', 2', 3', ...)

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Nucleosides & NucleotidesNucleoside: a compound that consists of D-ribose or 2-deoxy-D-ribose covalently bonded to a base by a -N-glycosidic bond. Lacks phosphate group.

Nucleotide: a nucleoside in which a molecule of phosphoric acid is esterified with an -OH of the monosaccharide, most commonly either the 3-OH or the 5-OH.

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b-N-Glycosidic BondIn nucleotides the pentose ring is attached to the nucleobase via N-glycosidic bond The bond is formed: to position N1 in pyrimidinesto position N9 in purines This bond is quite stable toward hydrolysis, especially in pyrimidinesBond cleavage is catalyzed by acidRelatively free rotation can occur around the N-glycosidic bond in free nucleotides

Continue. Name based on parent nucleoside with a suffix monophosphate.

Polymerization leads to nucleic acids.

Linkage is repeated (3,5-phosphodiester bond).*2-*

DNA structure- Part II

*2-*Levels of DNA structure

Levels of DNA structure 1structure: the order of bases on the polynucleotide sequence; the order of bases specifies the genetic code.

2structure: the three-dimensional conformation of the polynucleotide backbone.

3structure: supercoiling.

4structure: interaction between DNA and proteins

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DNA - 1 StructureDeoxyribonucleic acids (DNA) is a biopolymer that consists of a backbone of alternating units of 2-deoxy-D-ribose and phosphate.

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2-*The 3-OH of one 2-deoxy-D-ribose is joined to the 5-OH of the next 2-deoxy-D-ribose with bases by a phosphodiester bond.Continue.

2-*So, the Primary Structure is the sequence of bases along the pentose-phosphodiester backbone of a DNA molecule

Base sequence is read from the 5 end to the 3 end.

System of notation single letter (A,G,C,U and T).Continue.

2-* The backbone of DNA & RNA is hydrophilic.The hydroxyl gp (OH) of the sugar residues form hydrogen bonds with water.The Phosphate gp, with pka 2, are completely ionized and vely charged at pH 7. The ve charges are neutralized by ionic interactions with +ve charges on proteins, metal ions. A short nucleic acid containing 50 or fewer nucleotides is called an oligonucleotide, a longer is a polynucleotide.

*2-*the 2 hydroxyl is absent in DNA

Abbreviations of Nucleic Acid Sequences pA-C-G-T-AOH or pApCpGpTpA or pACGTA*2-*

Nucleotide Bases affect the three dimensional structure of nucleic acids The purine and pyrmidine bases are conjugated ring system. One result is that pyrmidines are planar molecules and purines are nearly planar with a slight pucker. Free purine and pyrimidine bases can exist in two or more forms called tautomers, depending on pH.*2-*A tautomeric shift occurs when a proton changes its position, resulting in a rare tautomeric form.

Continue. Standard and anomalous base-pairing arrangements that occur if bases are in the rare tautomeric forms. Base mispairings due to tautomeric shifts were originally thought to be a major source of errors in replication.Such structures have not been detected in DNA, and most evidence now suggests that other types of anomalous pairings are responsible for replication errors.*2-*

As a result of resonance, delocalized electrons in the conjugated ring rings are available to absorb UV light at 260nm. The chemical properties of the purine and pyrimidines give rise to two modes of interaction between bases:Hydrophobic stacking, the bases relatively insoluble in water at neutral pH.Base pairing which result from H-bonding .2-*Continue.As a result of resonance, all nucleotide bases absorb UV light.

Nucleotides play additional roles in cells Adenosine is a building block for some important enzyme cofactor, such as NAD+ and FAD. The presence of an adenosine component in a variety of cofactors enables recognition by enzymes that share common structural features.2-*FADNAD+

cAMP formed from ATP in a reaction catalyzed by adenylyl cyclase, is a common second messenger produced in response to hormones and other chemical signals*2-*Continue.

DNA - 2 StructureSecondary structure is the ordered arrangement of nucleic acid strands.

Double helix is a type of 2 structure of DNA molecules in which two anti-parallel polynucleotide strands are coiled in a right-handed manner about the same axis.

Structure based on X-Ray crystallography.

The molecule is stabilized by hydrophobic interactions in its interior and by hydrogen bonding between the complementary bases pairs A-T and G-C2-*

Base PairingBase pairing is complementary.A major factor stabilizing the double helix is base pairing by hydrogen bonding between T-A and between C-GT-A base pair comprised of 2 hydrogen bonds.G-C base pair comprised of 3 hydrogen bonds

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Bases are hydrophobic and interact by van der Waals interactions.In standard B-DNA, bases exposed to the minor groove come in contact with water.Many bases adopt a propeller-twist in which base pairing distances are less optimal but base stacking is more optimal and water is eliminated from minor groove contacts. The propeller twist of the base pairs results in purine-purine clash in the center of the helix. Because the purines are larger than the pyrimidine rings, they extend beyond the helical axis of DNA.Base stackingHydrophobic, van der Waals, and electrostatic interactions favor the alignment of bases in an aqueous solution or within a polynucleotide chain the un-stacked orientation is disfavored.

DNA attempts to reduce purine-purine clash in several ways:

A. The base pairs rotate less along the helix axis in the purine-pyrimidine sequences (lower average helical twist). They tend to rotate less in the pyrimidine-purine sequences (lower than average helical twist). The average helical twist was still very close to the 36 proposed by Watson and Crick.

Another way DNA minimizes the purine-purine clash is that it bends toward the minor grove or major groove to reduce the interaction.

C. Finally clashing base pairs could slide left or right toward the phosphodiester backbones to minimize the purine-purine interaction.2-*

Major and minor groovesThe major groove is large enough to accommodate an alpha helix from a protein. Regulatory proteins (transcription factors) can recognize the pattern of bases and H-bonding possibilities in the major groove.

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DNA adopts different helical forms2-*Nucleic acids are inherently flexible molecules. Numerous bonds in the sugar-phosphate backbone can rotate, and thermal fluctuation can lead to bending, stretching, and un-pairing of the two strands.

As a result, cellular DNA contains significant deviations from the Watson-Crick DNA structure, some or all of which may play important roles in DNA metabolism.

Generally, such structural variations do not affect the key properties of strand complementarity: antiparallel strands and the requirement for AT and GC base pairs.

Variation in the three-dimensional structure of DNA reflect three things:(2) DNA nucleotide conformation is affected by rotation about seven different bonds. Six of the bonds rotate freely; rotation about bond 4 is constrained by the sugar ring, giving rise to the sugar pucker.(1) The pentose rings in nucleosides and nucleotides can exist in four distinct puckered conformations.

The different possible confirmations of the deoxyribose. Rotation about the contiguous bonds that make up the sugar-phosphate backbone.Free rotation about the glycosidic bond.

Summary.

B-DNA (Watson-Crick Form) considered the physiological forma right-handed helix, diameter 1110 base pairs per turn (34) of the helix.

A-DNA (favored in environment with very low water content)a right-handed helix, but thicker than B-DNA.11 base pairs per turn of the helix has not been found in vivo.

Z-DNA occurs at high salt concentrations in polymers that have a sequence of alternating purines and pyrimidines a left-handed double helix.may play a role in gene expression.Z-DNA occurs in nature, usually consists of alternating purine-pyrimidine bases.Methylated cytosine found also in Z-DNA.

Continue.

In each case, the sugar-phosphate backbones wind around the exterior of the helix (red and blue), with the bases pointing inward. The same 25-base-pair DNA sequence is shown in all three forms. Differences in helical diameter can be seen in end-on views (top); differences in helical rise and groove shape are apparent in the side views (bottom). B-DNA, the most common form in cells, has a wide major groove and a narrow minor groove. A-form helices, common for RNA and certain DNA structures, are more compact than B-DNA. The major groove is deeper and the minor groove is shallower than in B-DNA. Z-DNA, which forms only under high salt conditions or with CG-rich DNA sequences, is left-handed, and its backbone has a zigzag pattern. It is less compact than B-DNA, with a very shallow major groove and a narrow and deep minor groove.

DNA - 3 StructureTertiary structure is the three-dimensional arrangement of all atoms of a nucleic acid; commonly referred to as supercoiling.

The term "supercoiling" means literally the coiling of a coil.

DNA is coiled in the form of a double helix.

Enzymes called topoisomerases or gyrases can introduce or remove supercoils.If there is no net bending of the DNA axis upon itself, the DNA is said to be in arelaxedstate. Negative supercoiling may promote cruciforms

Certain DNA Sequences Adopt Unusual Structures

Other sequence-specific DNA structures have been detected, within larger chromosomes, that may affect the function and metabolism of the DNA segments in their immediate vicinity. For example, certain repetitive sequences can bend the DNA helix in a distinct way.

This DNA bending helps certain proteinssuch as transcription factors, which promote the synthesis of mRNAsbind to their target DNA binding sites.

Regions of DNA where the two complementary strands have the same sequence when read in the 53 or the 35 direction occur relatively frequently in chromosomal DNA and are called palindromes.

Continue.

In language, a palindrome is a word, phrase, or sentence that is spelled identically when read either forward or backward. Two examples are ROTATOR and NURSES RUN. In biology, the term applies to double-stranded regions of DNA where one strands sequence is identical to its complement. for example, 5-GAATTC-3 is a palindrome because its complementary sequence is also 5-GAATTC-3. Palindromes are formed from adjacent inverted repeats, which can occur within one strand of DNA or over the two strands of the double helix

These sequences play important biological roles, such as:

Slowing or blocking protein synthesis by the ribosome-a process called translation attenuation .Forming recognition sites for restriction enzymes, which catalyze double-stranded DNA cleavage.

Continue.

DNA classes and sizes phage DNAHuman chromosome DNAPlasmid DNAE. Coli DNACircular DNA is a type of double-stranded DNA in which the 5 and 3 ends of each stand are joined by phosphodiester bonds.

GeneProteinWhat is in the middle??????

RNA Structure

In the early 1970s, Alexander Rich, Aaron Klug, and Sung-Hou Kim independently solved the structures of transfer RNAs, revealing how tRNAs carry the amino acids that are used in protein synthesis on the ribosomes.The wide-ranging functions of RNA reflect a structural diversity much richer than that observed in DNA molecules.

The single strand of RNA folds back on itself to form short base-paired or partially base-paired segments connected by unpaired regions. This property, called RNA secondary structure, enables RNA molecules to fold into many different shapes that lend themselves to many different biological functions.

Continue.

The greater structural variety in RNA relative to DNA reflects the three main chemical differences between the two polynucleotides: 1- The pentose (2-deoxyribose in DNA vs. ribose in RNA).2- The base composition (thymidine vs. uridine).3- The sugar pucker of the pentose (C-2 endo vs. C-3 endo).

Double-stranded RNAs do exist in nature, such as those that form the genomes of some viruses.In addition, some RNAs do not seem to form stable three-dimensional structures from local base-pairing interactions, e.g. mRNA. These RNAs may fold into three-dimensional structures only in the presence of bound proteins, forming complexes called ribonucleoproteins (RNPs).

Continue.

Most of the highly structured RNAs contain noncanonical base pairs and backbone conformations not observed in DNA.

In many cases, the 2-hydroxyl group on ribose, a chemical feature that distinguishes RNA from DNA, seems to be directly or indirectly responsible for these unique structural properties.

The presence of the 2 hydroxyl makes RNA vulnerable to hydrolysis, but it also allows for additional hydrogen bonding between segments of an RNA molecule.

As a result, RNA helices are more thermodynamically stable than are DNA helices of the same length and sequence.

RNAs Form Various Stable Three-Dimensional Structures

Base pairs other than canonical AU and CG pairs are common in RNAs, including A-A and G-U. In all cases, base pairs or single bases are most stable when stacked on top of one another in a helix.Divalent and monovalent metal ions (Mg2+, Ca2+, K+, and Na+) bind to specific sites in RNA and help shield the negative charge of the backbone, allowing parts of the molecule to pack more tightly together.

Continue.

Vicinal Hydroxyl Group Makes Difference !!

The vicinal OH groups of RNA are susceptible to nucleophilic attach leading to hydrolysis of the phosphodiester bond.

DNA is not susceptible to alkaline hydrolysis. RNA is alkali labile.

RNA is very similar to DNAChemically, RNA is very similar to DNA. There are some main differences:

1- RNA uses the sugar ribose instead of deoxyribose in its backbone.

It is because cytosine deaminates to form uracil in a finite rate in vivo. This would results in a mutation in the DNA:

So any U found in DNA will be corrected by a proofreads system. Thus DNA can not normally have U.Continue2- RNA uses the base Uracil (U) instead of Thymine (T). U is also complementary to A. But Why DNA Contains Thymine rather than Uracil?!

3- RNA tends to be single-stranded.

4- Functional differences between RNA and DNA.

DNA single function, RNA many functions according to their type.Example of types of RNA e.g: tRNA, mRNA, rRNA.Continue

The Central DogmaThe Flow of Information: DNA RNA Protein.

A gene is expressed in two steps: DNA is transcribed to RNA. Then RNA is translated into protein.

tRNA.rRNA.a vast number of other non-coding RNAs (ncRNAs).Many RNA molecules do not encode proteins

Of all RNA, transcribed in higher eukaryotes, 98% are never translated into proteins. Of those 98%, about 50-70% are introns. 4% of total RNA is made of coding RNA. The rest originate from non-protein genes, including rRNA, tRNA and a vast number of other non-coding RNAs (ncRNAs). Even introns have been shown to contain ncRNAs, for example snRNAs. It is thought that there might be order of 10,000 different ncRNAs in mammalian genomeHow big part of human transcribed RNAresults in proteins?

ncRNA genes have diverse and essential roles.May be relics of ancient RNA-based life.Many cellular machines contain RNA. Ribosome rRNA Spliceosome snRNAs (U1,U2,U4,U5,U6) Telomerase Telomerase RNAThe Other 98% of the Human Genome

RNA typesrRNA Participate in protein synthesissnRNA: RNA thatform part of thespliceosomeNon-coding RNA. Transcribed RNA with a structural, functional or catalytic rolemRNAsnRNA found in nucleolus, involved in modification of rRNA.tRNA Interface between mRNA &amino acidsmiRNA involvedregulation of expressionOther large RNAwith roles inchromotin structurestRNA- Small temporal RNA, with a role indevelopmental timingsiRNA- Small interfering RNA, Active molecules inRNA interference

RNA molecules are classified according to their structure and function

tRNA The smallest kind of the three RNAs. A single-stranded polynucleotide chain between 73-94 nucleotide residues. Intramolecular hydrogen bonding occurs in tRNA.

rRNArRNA: a ribonucleic acid 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. In both prokaryotes and eukaryotes, ribosomes consist of two subunits, one larger than the other.

mRNA

Single stranded and unstable.

Biosynthesis is directed by information encoded on DNA.

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

A ribonucleic acid that carries coded genetic information from DNA to ribosomes for the synthesis of proteins.

Present in cells in relatively small amounts and has a short-half life.

Eukaryotic mRNA Structure

Denaturation and renaturationChemical and thermodynamic importance of the DNA structure2-*

Properties of DNA- Denaturation

When DNA is heated to 80OC, its UV absorbance increases by 30-40% .With heating, noncovalent forces holding DNA strands together weaken and break. When the forces break, the two strands come apart in denaturation or melting.

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Denaturation of DNAAs strands separate, absorbance at 260 nm increases. Stacked base pairs in native DNA absorb less light . Increase is called hyperchromicity. Temperature at which DNA strands are denatured is the melting temperature or Tm. Melting Temperature (Tm) the temp. at which half of the helical structure is lost2-*Hyperchromic Effect a large increase in light absorbance at 260 nm occurring as double-helical DNA is melted (i.e. unwound).

Melting temperature and % G-CExamplesGC content of DNA has a significant effect on Tm with higher GC content meaning higher Tm.2-* Upon denaturation, the H-bonds between the base pairs of a native nucleic acid are replaced by energetically equivalent hydrogen bonds between the bases and water

ContinueIn addition to heat, DNA can be denatured by:Organic solvents.High pH.Low salt concentration.

GC content also affects DNA densityDirect, linear relationship2-*

DNA RenaturationAfter two DNA strands separate, under proper conditions the strands can come back togetherProcess is called annealing or renaturationThree most important factors:Temperature best at about 25 C below Tm.DNA Concentration within limits higher concentration better likelihood that 2 complementary will find each other.Renaturation Time as increase time, more annealing will occur2-*

SummaryGC content of a natural DNA can vary from less than 25% to almost 75%.GC content has a strong effect on physical properties that increase linearly with GC content.Melting temperature, the temperature at which the two strands are half-dissociated or denatured.Density.Low ionic strength, high pH and organic solvents also promote DNA denaturation.2-*

HybridizationAnnealing of complementary DNA (hybrid duplex) from different species at 65.2-*

Continue Hybridization is a process of putting together a combination of two different nucleic acids.

Strands could be 1 DNA and 1 RNA.

Also could be 2 DNA with complementary or nearly complementary sequences.2-*

DNA Sizes DNA size is expressed in 3 different ways:Number of base pairs .Molecular weight 660 is molecular weight of 1 base pair.Length 33.2 per helical turn of 10.4 base pairs. Measure DNA size either using electron microscopy or gel electrophoresis. 2-*

SummaryNatural DNAs come in sizes ranging from several kilobases to thousands of megabases.

The size of a small DNA can be estimated by electron microscopy.

This technique can also reveal whether a DNA is circular or linear and whether it is supercoiled.

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*****FIGURE 81 Structure of nucleotides. (a) General structure showing the numbering convention for the pentose ring. This is a ribonucleotide.In deoxyribonucleotides the OH group on the 2 carbon (in red) is replaced with H. (b) The parent compounds of the pyrimidine and purine bases of nucleotides and nucleic acids, showing the numbering conventions.*************************************