Elements of BiotechnologyUnit 2

B.Tech Biotechnology II

Basic concepts of Genes, DNA & RNA

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BREAKTHROUGH DISCOVERY

• In 1953, James Watson and Francis Crickdiscovered the double helical structure of the DNA molecule

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DNA

A purine always links with a pyrimidine base to maintain the structure of DNA.Adenine ( A ) binds to Thymine ( T ), with two hydrogen bonds between them.Guanine ( G ) binds to Cytosine ( C ), with three hydrogen bonds between them.

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Nucleoside & Nucleotide, the

fundamental building block of DNA

glycosidic bond

phosphoester bond

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Ribose

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• Nucleotides have three characteristic components:

• (1) a nitrogenous (nitrogen-containing) base, (2) a pentose, and (3) a phosphate.

• The molecule without the phosphate group is called a nucleoside.

• The nitrogenous bases are derivatives of two parent compounds, pyrimidine and purine.

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DNA & RNA - Nucleotide Bases

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Nucleotide

adenine

deoxyribose

PO4

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Nucleotides

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CHEMICAL AND PHYSICAL PROPERTIES OF DNA

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PHYSICAL PROPERTIES OF DNA

• DNA Stores Genetic Information• Many lines of evidence show that DNA bears

genetic information. In particular, the Avery-• MacLeod-McCarty experiment showed that

DNA isolated from one bacterial strain can enter and transform the cells of another strain, endowing it with some of the inheritable characteristics of the donor. The Hershey-Chase experiment showed that the DNA of a bacterial virus, but not its protein coat, carries the genetic message for replication of the virus in a host cell. 14

DNA Is a Double Helix

• Putting together much published data, Watson and Crick postulated that native DNA consists of two antiparallel chains in a right-handed double-helical arrangement. Complementary base pairs, A=T and G C, are formed by hydrogen bonding within the helix. The base pairs are stacked perpendicular to the long axis of the double helix, 3.4 Å apart, with 10.5 base pairs per turn.

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• DNA Sequences Adopt Unusual Structures

• A number of other sequence-dependent structural variations have been detected within larger chromosomes that may affect the function and metabolism of the DNA segments in their immediate vicinity.

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• A rather common type of DNA sequence is a palindrome.

• A palindrome is a word, phrase, or sentence that is spelled identically read either forward or backward; two examples are ROTATOR and NURSES RUN.

• The term is applied to regions of DNA with inverted repeats of base sequence having twofold symmetry over two strands of DNA

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Such sequences are self-complementary within each strand and therefore have the potential to form hairpin or cruciform (cross-shaped) structures. When the inverted repeat occurs within each individual strand of the DNA, the sequence is called a mirror repeat.Mirror repeats do not have complementary sequences within the same strand and cannot form hairpin or cruciform structures.

A, B and Z DNA

• A form – favored by RNA

• B form – Standard DNA double helix under physiological conditions

• Z form – laboratory anomaly, – Left Handed– Requires Alt. GC– High Salt/ Charge

neutralization

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• Four DNA strands can also pair to form atetraplex (quadruplex), but this occurs readilyonly for DNA sequences with a very highproportion of guanosine residues.

• The guanosine tetraplex, or G tetraplex, isquite stable over a wide range of conditions.

• H-DNA, is found in polypyrimidine orpolypurine tracts that also incorporate a mirrorrepeat. A simple example is a long stretch ofalternating T and C residues. The H-DNAstructure features the triple-stranded formillustrated in Figure. Two of the three strandsin the H-DNA triple helix contain pyrimidinesand the third contains purines. 22

CHEMICAL PROPERTIES OF DNA

• ABSORPTION• The bases in DNA absorb ultraviolet light at

the wavelength of 260 nm• This absorption can be monitored using a

spectrophotometer• This is one method used to figure the

concentration of DNA in solution• The more DNA present, the higher the

absorption

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• DENSITY• Density can be measured by CsCl-density

ultracentrifugation• can be used to estimate G+C content• GC base pairs are more dense than AT base

pairs• Density studies show the existence of satellite

DNA

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• DENATURATION• DNA is considered denatured when the double

stranded DNA molecule is converted into two single stranded molecules

• As thermal energy increases, the frequency of hydrogen bonds breaking between the molecules increases

• G-C base pairs are held together by three hydrogen bonds (A-Ts by two) and it therefore takes more energy (higher temperatures) to separate molecules with high GC contents

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DNA can Form Hybrids

• The ability of two complementary DNA strands to pair with one another can be used to detect similar DNA sequences in two different species or within the genome of a single species.

• Hybridization techniques can be varied to detect a specific RNA rather than DNA. The isolation and identification of specific genes and RNAs rely on these hybridization techniques. Applications of this technology make possible the identification of an individual on the basis of a single hair left at the scene of a crime or the prediction of the onset of a disease decades before symptoms appear

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DNA hybridization.

Two DNA samples to be compared are completely denatured by heating. When the two

solutions are mixed and slowly cooled, DNA strands of each sample associate with their

normal complementary partner and anneal to form duplexes. If the two DNAs have

significant sequence similarity, they also tend to form partial duplexes or hybrids with each

other: the greater the sequence similarity between the two DNAs, the greater the number of

hybrids formed. Hybrid formation can be measured in several ways.

One of the DNAs is usually labeled with a radioactive isotope to simplify the

measurements.

Nucleotides and Nucleic Acids Undergo Nonenzymatic Transformations

• Purines and pyrimidines, along with the nucleotides of which they are a part, undergo a number of spontaneous alterations in their covalent structure.

• The rate of these reactions is generally very slow, but they are physiologically significant because of the cell’s very low tolerance for alterations in its genetic information.

• Alterations in DNA structure that produce permanent changes in the genetic information encoded therein are called mutations, and much evidence suggests an intimate link between the accumulation of mutations in an individual organism and the processes of aging and carcinogenesis.

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• Hydrophobicity of solvent• Hydrophobic substances will allow the bases in

DNA to dissolve into the solvent• Whereas hydrophilic substances will keep the

bases of DNA stacked upon one another in the orientation that most favors hydrogen bonding between DNA strands

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• pH• Acidic pH cause breakage of phosphodiester

bonds between nucleotides and breakage of the N-glycosidic bond between the sugar and purine bases

• Alkali - Above pH 11.3, all hydrogen bonds are disrupted and the DNA is totally denatured

• Salts will stabilize the DNA double helix

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• Electrophoresis• DNA has a negative charge that is proportional to

its size• This is due to the negatively charged phosphates

in the sugar-phosphate backbone• If DNA is placed in an electrical field it will migrate

towards the positive electrode (the cathode)• smaller pieces will migrate faster than larger pieces• Larger pieces have trouble squeezing through the

gel matrix and are hence retarded while smaller pieces migrate easier

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• Type of gels• Agarose is used to separate fairly large DNA

molecules– 5 million to a few thousands base pairs

• Polyacrylamide is used to separate small pieces of DNA– 2 to several hundred base pairs

• The size of DNA is estimated by comparing its migration through the gel to DNA molecules of known size

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RNA

Three major classes of RNA:

Difference between RNA & DNARNA DNA

RNA nucleotides contain ribose sugar DNA contains deoxyribose

RNA has the base uracil DNA has the base thymine

presence of a hydroxyl group at the 2' position of the ribose sugar.

Lacks of a hydroxyl group at the 2' position of the ribose sugar.

RNA is usually single-stranded DNA is usually double-stranded33

mRNA

• Transcripts of structural genes.

• Encode all the information necessary for the

synthesis of a polypeptide of protein.

• Intermediate carrier of genetic information;

deliver genetic information to the cytoplasm.

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mRNA to Amino Acid Dictionary

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tRNA

RNA molecules 70- 100 nucleotide long.

The secondary structure of the tRNA

resembles a D loop, anticodon loop,

and T loop and the acceptor stem.

Carry correct amino acids to their

position along the mRNA template to be

added to the growing polypeptide chain.

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rRNA

• The central component of the

ribosome.

• Ribosome; factory for protein

synthesis; composed of ribosomal

RNA and ribosomal proteins.

• rRNA provides a mechanism for

decoding mRNA into amino acids.

• rRNA interact with the tRNAs during translation byproviding peptidyl transferase activity.

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Biological roles of RNA

1. RNA is the genetic material of some viruses

2. RNA functions as the intermediate (mRNA)

between the gene and the protein-synthesizing

machinery.

3. RNA functions as an adaptor (tRNA) between

the codons in the mRNA and amino acids.

4. RNA serves as a regulatory molecule, which

through sequence complementarity binds to,

and interferes with the translation of certain

mRNAs.

5. Some RNAs are enzymes that catalyze essential

reactions in the cell (RNase P ribozyme, large

rRNA, self-splicing introns, etc).38

Pseudoknots are complex structure

resulted from base pairing of

discontiguous RNA segments

Figure 6-32 Pseudoknot.

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Structure (1): RNA chains fold back on

themselves to form local regions of double

helix similar to A-form DNA

RN

A S

TR

UC

TU

RE

(2)

hairpin

bulge

loop

RNA helix are the base-paired segments between short stretches of complementary sequences, which adopt one of the various stem-loop structures

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Organization of DNA in eukaryotes

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• Genome

Gene• Is the basic units of

inheritance; it is a segmentwithin a very long strand ofDNA with specificinstruction for theproduction of one specificprotein.

• Genes located onchromosome on it's place orlocus.

Genome and Gene

• Totality of genetic information of an organism.• Encoded in the DNA (for some viruses, RNA).

Chargaff’s rule of equivalance

A=T and G=C

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Chargaff's rule

• Chargaff's rules state that DNA fromany cell of all organisms should have a 1:1ratio (base Pair Rule)of pyrimidine and purine bases and, morespecifically, that the amount of guanine isequal to cytosine and the amountof adenine is equal to thymine.

• They were discovered by Austrianchemist Erwin Chargaff

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• Deoxyribonucleic acid (DNA) is the genetic material found in the chromosomes of all animals and plants.

• It is made up of only four types of organic nitrogenous bases: adenine (A), guanine (G), thymine (T) and cytosine (C).

• Of these, A and G are the purines and T and C are the pyrimidines

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• Chargaff gave the base pairing rule or the rule of base equivalence which states that only one purine can combine with one pyrimidine.

• That means A can combine with T and G with C.

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Experiment

• Chargaff and his students collected numerous DNAsamples for various organisms. Using the fairly newtechnique of paper chromatography, Chargaff and hisassociates proceeded to separate DNA.

• The DNA that they collected was subjected to acid.The acid would then hydrolyze the phospodiesterbonds as it would cause a nucleophilic attack on thebond and result in the backbone breaking up. Once thephosphodiester bonds were broken then the individualnucleotides would then be separated and be free toanalyze. Ultraviolet spectrophotometry was used toanalyze the exact amounts of bases that were present inthe DNA sample.

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Relative proportions (%) of bases in DNA

Organism %A %G %C %T A/T G/C %GC %AT

φX174 24 23.3 21.5 31.2 0.77 1.08 44.8 55.2

Maize 26.8 22.8 23.2 27.2 0.99 0.98 46.1 54

Octopus 33.2 17.6 17.6 31.6 1.05 1 35.2 64.8

Chicken 28 22 21.6 28.4 0.99 1.02 43.7 56.4

Rat 28.6 21.4 20.5 28.4 1.01 1 42.9 57

Human 29.3 20.7 20 30 0.98 1.04 40.7 59.3

Grasshopper 29.3 20.5 20.7 29.3 1 0.99 41.2 58.6

Sea Urchin 32.8 17.7 17.3 32.1 1.02 1.02 35 64.9

Wheat 27.3 22.7 22.8 27.1 1.01 1 45.5 54.4

Yeast 31.3 18.7 17.1 32.9 0.95 1.09 35.8 64.4

E. coli 24.7 26 25.7 23.6 1.05 1.01 51.7 48.3

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Circular and super helical DNA

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• Enzymes called topoisomerases can take apart a circular DNA, introduce additional twists into it, and then reseal the structure.

• Adding twists to circular DNA introduces tension into the molecule.

• The extra tension in circular DNA (or in linear DNA whose ends are anchored to prevent tension from being released) usually causes the molecule to writhe to alleviate the tension. Like an overwound rubber band, the circular DNA assumes a new shape, called a supercoil.

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Relaxed and supercoiled DNA molecules

• Supercoiling can be positive (additional twists added beyond the normal amount for linear DNA) or negative (reduced numbers of twists compared to linear DNA).

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• The unstrained circle contains the same number of twists as linear DNA. It is under no superhelicaltension.

• To make the strained circle, one twist was removed (compared to linear DNA) and the resulting circular DNA is strained because it has the same number of base pairs (105), but fewer numbers of turns (twists). Thus, the strained circle has a higher number of base pairs per turn than the unstrained circle.

• To relieve the strain, the strained molecule can introduce another superhelical turn within itself, called a writhe.

• After the writhe, the number of twists (turns) is 10 again so the number of base pairs per turn is 10.5 again, too.

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• The linking number (L) is simply sum of the number of twists (T) and writhes (W) of a molecule:

• L = T + W• Consequently, the change in the linking number

is also equal to the change in the twists and writhes for a molecule:

• ΔL = ΔT + ΔW• The superhelical density is defined as Δ L/L0,

where L0 is the linking number of the DNA in its unstrained (relaxed state).

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• Many naturally occurring DNA molecules have superhelicaldensities of about -0.06. To get an idea of what this means, consider a hypothetical DNA molecule of 10,000 bp, which is in the "classical" B form, with 10.0 bp/turn. Then L0 is 10,000 bp/(10.0 bp/turn), or 1000 turns.

• Each DNA strand crosses the other 1000 times in the relaxed circle. If the topoisomerase gyrase twisted the molecule to a superhelical density of -0.06, then L = -0.06 L0, or L = -60. This change could be accommodated, for example, by the helix axis writhing about itself 60 times in a left-hand sense, which would correspond to W = -60, T = 0; the molecule would have 60 left-hand superhelical turns.

• Alternatively, the twist of the molecule could change so that it had 940 turns in 10,000 bp (T = 940) or 10,000/940 = 10.64 bp/turn. This would correspond to W = 0, T = -60. Although any combination of T and W that sums to -60 could occur, real molecules release strain mainly by writhing into superhelical turns, because it is easier to bend long DNA than it is to untwist it.

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THE TOPOLOGICAL PROPERTIES OF DNA HELP US TO EXPLAIN

– DNA COMPACTING IN THE NUCLEUS– UNWINDING OF DNA AT THE

REPLICATION FORK– FORMATION AND MAINTENANCE OF THE

TRANSCRIPTION

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References

• Images references:

1-2 Lehninger Principles of biochemistry by Nelson and Cox

• Reading references:

• Gene cloning and DNA analysis by TA Brown

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