NUCLEOTIDES

There are 8 common varieties of nucleotides, each consists of 3 part: A nitrogen based A pentose sugar, either deoxyribose or ribose At least one phosphate group

NITROGEN BASED Nucleotides can divided into 2 class or group Cytosine (C), thymine (T) and Uracil (U) have single rings and they are pyrimidinine adenine guanine Adenine (A) and guanine (G) are double-ringed molecule of class called purine

Purine form bonds to a five-carbon sugar (a pentose) via their N9 atoms Pyrimidines do so through their N1 atoms

PENTOSE SUGAR In ribonucleotides, the pentose is ribose In deoxyribonucleotide (or deoxynucleotides) the sugar is 2’-deoxyribose – the carbon at position 2’ lacks a hydroxyl group

In ribononucleotide, one or more phosphate groups is bonded to atom C3’ or atom C5’ of the pentose to form a 3’-nucleotide or a 5’-nucleotide. When phosphate group is absent, the compound is known nucleoside. Ribonucleotides are found in RNA (ribonucleic acid) Deoxynucleotides are found in DNA (deoxyribonucleic acid)

A, G, C found as both RNA and DNA U is found as RNA T is found as DNA

Definitions DNA stands for deoxyribonucleic acid. It is the genetic code molecule for most organisms. RNA stands for ribonucleic acid. RNA molecules are involved in converting the genetic information in DNA into proteins. In retroviruses, RNA is the genetic material.

Nucleotide and Nucleotide derivatives The bulk of nucleotides in any cell are found in polymeric form, as either DNA and RNA Primary function are information storage and transfer Free nucleotides and nucleotides derivatives perform an enormous variety of metabolic functions and not related to management of genetic information

The best known is ATP (adenosine triphosphate) contains adenine, ribose and triphosphate group ATP is the principal, short –term, recyclable energy supply for cells. When the phostphate bonds of ATP are broken, a significant amount of energy is release. More energy is release from phosphate bonds than is release from most other covalent bonds. For this reason, the phosphate-phosphate bonds of ATP are known as high-energy bonds Energy is release when ATP is convert to ADP, and when phosphate is remove from ADP to form AMP, though the latter reaction is not as common in cells Energy release from the phosphate bonds of ATP is used for important life-sustaining activities, such as synthesis reactions, locomotion, and transportation of substance into and out of cells. Cells also use ATP as a structural molecule in formation of coenzymes.

Nucleic acid structure

Segment of DNA Chain 5’-end guanine thymine cytosine 3’-5’ link 3’-end

Nucleic acid structure Nucleotides can be joined to each other to form RNA and DNA. The nucleic acids are chains of nucleotides whose phosphates bridge the 3' and 5' positions of neighboring ribose units The phosphates of these polynucleotides are acidic, so at physiological pH, nucleic acids are polyanions. The linkage between individual nucleotides is known as a phosphodiester bond. Each nucleotide that has been incorporated into the polynucleotide is known as a nucleotide residue. The terminal residue whose C5' is not linked to another nucleotide is called the 5' end The terminal residue whose C3' is not linked to another nucleotide is called the 3' end. By convention, the sequence of nucleotide residues in a nucleic acid is written, left to right, from the 5' end to the 3' end.

Or: pdGpdTpdC Or: pd(GTC) RNA abbreviations lack the d (for deoxy)

DNA and RNA chains are abbreviated using a structure where vertical lines represent the sugars, diagonal lines with P at the midpoint represent the 3,5-phosphodiester bonds, and horizontal lines the ends of the chain. The structures are always written with the 5’ end to the left. Single letter abbreviations are also used.

The base composition of DNA Though there appear to be no rules governing the nucleotide composition of typical RNA molecules, DNA has equal numbers of adenine and thymine residues (A = T) and equal numbers of guanine and cytosine (G = C). These relationships, known as Chargaff’s rules. The significance of Chargaff's rules was not immediately appreciated, but we now know that the structural is for the rules derives from DNA's double-stranded nature.

The double helix Watson-Crick structure: Two polynucleotide chains wind around a common axis to form a double helix. The two strands of DNA are antiparallel, but each forms a right-handed helix. The bases occupy the core of the helix and sugar-phosphate chains run along the periphery, thereby minimizing the repulsions between charged phosphate groups. The surface of the double helix contains two grooves of unequal width: the major and minor grooves.

The double helix Each base is hydrogen bonded to a base in the opposite strand to form a planar base pair. Each adenine residue must pair with a thymine residue and vice versa, and each guanine residue must pair with a cytosine residue and vice versa. These hydrogen­bonding interactions, a phenomenon known as complementary base pairing, result in the specific association of the two chains of the double helix.

Single stranded nucleic acid Single-stranded DNA is rare In contrast, RNA occurs primarily as single strands, which usually form compact structures rather than loose extended chains A RNA strand-which is identical to a DNA strand except for the presence of OH groups and the substitution of uracil for thymine-can base-pair with a complementary strand of RNA or DNA. As expected, A pairs with U or T in DNA, and G with C. Base pairing often occurs intramoleculy giving rise to stem-loop structures or, when loops interact with each other, to more complex structures.

The DNA helix The geometry of DNA The biologically most common form of DNA is known as B-DNA, - structural features first noted by Watson and Crick together with Rosalind Franklin and other. Double-helical DNA can assume several distinct structural depending on the solvent composition and base sequence. The major structural variants of DNA are A- and Z-DNA. Under dehydrating conditions, B-DNA undergoes a reversible conformational change to A-DNA which forms a wider and flatter right-handed helix than does B-DNA. Key to structure. Structural features of ideal A-, B- and Z-DNA.

B DNA segment Sugar-phosphate backbone Chain 2 Chain 1 Hydrogen bonded base pairs in the core of the helix

B DNA: 2 Major groove Outside diameter, 2 nm Interior diameter, 1.1 nm Length of one turn of helix is 3.4 nm and contains 10 base pairs. Interior diameter, 1.1 nm Minor groove

A DNA and Z DNA A second form of DNA is the A form. It has 11 base pairs per turn of the helix and the bases lie at an angle of about 20o relative to the helix axis. It, too, is a right hand double helix. A third form of DNA is the Z form. It is a left handed helix. A picture of A DNA is on the next slide.

A DNA segment Base pairs not perpendicular to helix axis. 11 pairs per turn.

Force stabilizing nucleic acid structure - DNA Denaturation and renaturation When a solution of duplex DNA is heated above a characteristic temperature, its native structure collapses and its two complementary strands separate and assume randam conformations. The stability of the DNA double helix, and its Tm (melting temperature) depends on several factors including - the nature of the solvent, - the identities and concentration of the ions in solution, -and the pH. Tm also increases linearly with the mole fraction of G.C base pair, although this is not, as one might expect, entirely because G.C base pairs contain one more hydrogen bond than A.T base pair.

Base pairing Base pairing is apparently a “glue” that holds together double-stranded nucleic acids. Only Watson-crick pairs occur in the crystal structure of self-complementary oligonucleotides. Other hydrogen-bonded base pairs with reasonable geometric are known. Observation indicate that Watson-crick geometry is the most stable mode of base pairing in the double helix, even though non Watson-crick base pairs are theorically possible. Hydrogen bonding contribute little to the stability of nucleic acid structure. This is because, on denaturation, the hydrogen bonds between the base pair of native nucleic acid are replaced by energetically similar hydrogen bonds between the bases and water. Only types of forces must therefore play an important role in stabilizing nucleic acid structure.

Base stacking and hydrophobic interaction Stacking interactions are a form of van der waals interaction. Interaction between stacked G and C bases are greater than those between stacked A and T bases, which largely accounts for the greater thermal stability of DNAs with a high G+C content. Note also that differing sets of base pairs in a stack have different staking energies. Thus, the stacking energy of a double helix is sequence-dependent.

RNA RNA is stabilizing by the same force that stabilizing DNA. In fact RNA may be even more rigid than DNA to presence of greater number of water molecule that form hydrogen bond to the 2’-OH group of DNA. RNAs may contain double-stranded segments

Fractionation of nucleic acids Most of common procedures for isolating and characterizing protein, often with some modification, are also used to fractionate nucleic acid according to size, composition and sequence. Some of the most useful nucleic acid fractionation procedure are Chromatography Electrophoresis Ultracentrifugation

Chromatography Many of the chromatographic techniques that are used to separate proteins also apply to nucleic acids. Hydroyapatite, a form of calcium phosphate is particularly useful in the chromatographic purification and fractionation of DNA. Double stranded DNA binds to hydroyapatite more tightly than do most other molecules. DNA can be rapidly isolated by passing a cell lysate through a hydroyapatite column, washing the column with a low concentration of phosphate buffer to release only the RNA and protein, and then eluting the DNA with a concentrated phosphate solution. Affinity chromatography is used to isolate specific nucleic acids. For example, most eukaryotic messenger RNAs (mRNAs) have a poly (A) sequences or cellulose to which poly (U) is covalently attached. The poly(A) sequences specifically bind to the complementary poly(U) in high salt and low temperature and can later be released by altering these condition.

Electrophoresis Gel electrophoresis is a technique for separating molecules (including fragments of nucleic acids) by size, shape, and electrical charge. The technique involves drawing DNA molecules, which have an overall negative charge, through a semisolid gel by an electric current toward the positive electrode within an electrophoresis chamber. The gel is typically composed of a purified sugar component of agar called agarose. Smaller DNA fragments move faster and farther than larger ones. Saiz of a fragments detrmined by comparing the distance it travels to the distances traveled by standard DNA fragment of known size. In genetic engineering, scientists use the technique to isolate fragments of DNA molecules that can then be inserted into vectors, multiplied by PCR, or preserved in a gene library.

Southern blot The Southern blot technique begins with the procedures of gel electrophoresis just described, but allows researchers to stabilize specific DNA sequences and then localize them using DNA dyes or probes. Once the DNA fragments have been separated by size, the liquid in the electrophoresis gel is blotted out, the DNA is denatured with NaOH, and its single strands are transferred and bonded to anitrocellulose membrane. The Southern blot is used for genetic fingerprinting, diagnosis of infectious disease, and other purposes. Also to demonstrate the incidence and prevalence in an environmental sample of archaea, bacteria, and viruses, particularly those that cannot be cultured. Northen blot is a similar technique used to detect specific RNA molecule. If the gel separates DNA and the DNA is detected with a DNA probe, it is called a Southern blot. If RNA is separated on the gel and then detected by a DNA probe, it is a Northen blot. A Western blot uses specific antibodies to detect specific protein molecules on a blot of a protein gel. In the Western blot, the role of the DNA probe is filled by an antibody that recognized a specific protein.

Southern blot technique

DNA Microarrays DNA microarrays consist of molecules of single-stranded DNA that has been immoblized on glass slides, silicon chips, or nylon membranes. Single strands of fluorescently-labeled DNA in a sample washed over an array adhere only to locations on the array where there are complementary DNA sequences. DNA microarrays can be used in a number of ways including monitoring gene expression, diagnosing infection, and identifying organisms in an environmental sample.

Ultracentrifugation Equilibrium density gradient ultracentrifugation in CsCl is one of the most commonly used DNA separation procedures. The buoyant density of double stranded Cs+ DNA depends on its base composition, with DNAs of higher G + C content having a greater density. Single stranded DNA is ~0.015g cm-3 denser than the corresponding double stranded DNA, so the two can be separated by equilibrium density gradient ultracentrifugation. Circular DNAs that are supercoiled to different extents.