DNA structure determination

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• Franklin collected x-ray diffraction data (early 1950s) that indicated 2 periodicities for DNA: 3.4 Å and 34 Å.

• Watson and Crick proposed a 3-D model accounting for the data.

CELL STRUCTURE AND BIOMOLECULES

Molecular biology- Energy based flow of information from DNA to RNA to proteins and enzymes i.e. from Hypo pituitary access to hormones to homeostatic etc.

The mother of all biomolecules

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1ffk

Large subunit of the ribosome(proteins at least)

Chromatin Structure

INACTIVE &ACTIVE CHROMATIN

• Active (transcriptionally active)differ from from inactive region.

• DNA in active region contain large

regions(100,000bp)prone to digestion by Dnase- I (single strand cut)

MAJOR AND MINOR GROOVES

• MINOR– EXPOSES EDGE FROM WHICH C1’ ATOMS

EXTEND

• MAJOR– EXPOSES OPPOSITE EDGE OF BASE PAIR

• THE PATTERN OF H-BOND POSSIBILITIES IS MORE SPECIFIC AND MORE DISCRIMINATING IN THE MAJOR GROOVE

DNA structure

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Fig. 8-15

•DNA consists of two helical chains wound around the same axis in a right-handed fashion aligned in an antiparallel fashion.

• There are 10.5 base pairs, or 36 Å, per turn of the helix.

• Alternating deoxyribose and phosphate groups on the backbone form the outside of the helix.

• The planar purine and pyrimidine bases of both strands are stacked inside the helix.

Much of Genome is not TRANSCRIBED

• The entire human haploid genome contain sufficent DNA to code for app. 1.5 million genes.

• Human genome encodes less than 100,000 proteins ie 1% of human genome.

• 24% of genome as Introns

More than half DNA of Eukaryotic is non Repetitive Sequences.

• In humans 10,000 to 15000 . genes are expressed.

• Different combination of genes are expressed in each tissue,of course and how this is accomplished in one of the major unanswered question in biology.

30% of Human Genome has Repetitive sequences

• A.Highly repetitive: 5-500 bp length repeated many times.(these seq. are transcriptionaly inactive)may play role in structure of ch.

• B. Moderately repetitive: 106 copies per haploid genome are not clustered but are interspred with unique seq.

• C. Microsatelite repeat seqs.2-6bp repeated up to 50 times(AC=TG)AC repeat seqs. Are50000-100000 locations in genome.

TELOMERE

• The ends of each chromosome contains structures called TELOMERES

• Telomere consist of short repeat TG sequences.(5’-TTAGGG-3’)can run for kb.

• TELOMERASE ( RNA+RNA dependent DNA polymerasesor{ Reverse transcriptase} is responsible for telomere synthesis) & maintains length of telomere.

Cont………………………….• Genome of Prokaryotes are

circular.human &Eukaryotes is linear as result the lagging strand has incomplete 51 end. Each round of replication would shorten the chromosome.Telo=End.

• How are repeated sequences generated the enzyme TELOMERASE performs this function.

• The protein component of telomerases,hence it acts as reverse transcriptase.that carries its own template.

• Telomerase levels are raised in cancer cells.

DNA• Two helical polynucleotide chains are

coiled around a common axis.• The chains run in opposite directions.

(Template and Coding)• Sugar-Phosphate backbones are on

the outer side and Purine &Pyrimidene bases lie on the inside of helix.

• The bases are prepandicular to the helix axis. (10 bases per turn of Helix)

• Diameter of helix is 20 Ao.

Cont………………………..• In between large regions there are

shorter streches (100-300)which are more sensative to Dnase-I These sites provides a sight for transcription.(euchromatin)

• Transcriptionally inactive ch. Is densly packed.(Hetrochromatin)

A-DNA

• RIGHT-HANDED HELIX• WIDER AND FLATTER THAN B-DNA• 11.6 BP PER TURN• PITCH OF 34 A

AN AXIAL HOLE

• BASE PLANES ARE TILTED 20 DEGREES WITH RESPECT TO HELICAL AXIS– HELIX AXIS PASSES “ABOVE” MAJOR GROOVE DEEP MAJOR AND SHALLOW MINOR GROOVE

• OBSERVED UNDER DEHYDRATING CONDITIONS

B-DNA• B-DNA Right handed double helix.• Supercoils store energy.• Supercoiled DNA is prefered form.• DNA of E.coli is1.4mm, 4.7million bp.• Eukaryotes contain more than ten

times of DNA as compaired to prok.

Z-DNA• A LEFT-HANDED HELIX• SEEN IN CONDITIONS OF HIGH SALT

CONCENTRATIONS– REDUCES REPULSIONS BETWEEN CLOSEST

PHOSPHATE GROUPS ON OPPOSITE STRANDS (8 A VS 12 A IN B-DNA)

• IN COMPLEMENTARY POLYNUCLEOTIDES WITH ALTERNATING PURINES AND PYRIMIDINES– POLY d(GC) · POLY d(GC)– POLY d(AC) POLY d(GT)

• MIGHT ALSO BE SEEN IN DNA SEGMENTS WITH ABOVE CHARACTERISTICS

B,A and Z DNA

10/10/0538Fig. 8-19

• B form - The most common conformation for DNA.

• A form - common for RNA because of different sugar pucker. Deeper minor groove, shallow major groove

• A form is favored in conditions of low water.

• Z form - narrow, deep minor groove. Major groove hardly existent. Can form for some DNA sequences; requires alternating syn and anti base configurations.

36 base pairsBackbone - blue;Bases- gray

DNA strands

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Fig. 8-16

• The antiparallel strands of DNA are not identical, but are complementary.

• This means that they are positioned to align complementary base pairs: C with G, and A with T.

• So you can predict the sequence of one strand given the sequence of its complement.

• Useful for information storage and transfer!

• Note sequence conventionally is given from the 5' to 3' end

Discovering the structure of DNA

• DNA = Deoxyribose nucleic acid

• Made out of sugars (deoxyribose), phosphates

and nitrogen bases

Difference betweenDNA and RNA

• Sugar: DNA;deoxyribose.

• RNA; Ribose.

• DNA: A,T,G,C.: RNA;A,U,G,C.

• DNA Double,RNA single stranded.

• DNA A=T,G=C, RNA; U=A,,G=C not.

• RNA can be hydrolysed by Alkali,DNA not.

HISTONES:

• H1• H2A• H2B• H3• H4

Acetylation linked with replication,H1 with condensation,DNA repair,transcription repression,methylation of histones activation and repression of gene.

HISTONES• Most abundant basic chr.protein. • H1Loosly bound to chr,&not not

necessary,but adjecent nucleosome is joined by H1, nucleosome

• Nucleosome contain four types of histones.H2A,H2B,H3 and H4

• Function identical in all eukaryotes.• Four core Histones are subject to 6

types of covalent modifications

HISTONES• Acetylation,Methylation,Phosphory

lation,ADP-ribosylation,monoubiquitylation and Sumoylation.

• H3,H4 form tetramer• h2A &H2B form Dimer.Under

physiologic conditions olig.associate & form octamer.

• The amino tail(His) is available co-valent modifications

• One octamer has 1.75 turns of DNA (146bp)

The mother of all biomolecules

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Large subunit of the ribosome(proteins at least)

Z-DNA• 12 (W-C) BASE PAIRS PER TURN• A PITCH OF 44 DEGREES• A DEEP MINOR GROOVE• NO DISCERNIBLE MAJOR GROOVE• REVERSIBLE CHANGE FROM B-DNA TO Z-DNA

IN LOCALIZED REGIONS MAY ACT AS A “SWITCH” TO REGULATE GENE EXPRESSION– ? TRANSIENT FORMATION BEHIND ACTIVELY

TRAN-

SCRIBING RNA POLYMERASE

FORCES THAT STABILIZE NUCLEIC ACID STRUCTURES

• SUGAR-PHOSPHATE CHAIN CONFORMATIONS

• BASE PAIRING• BASE-STACKING,HYDROPHOBIC • IONIC INTERACTIONS

DNA TOPOLOGY

• 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 BUBBLE• MANAGING THE SUPERCOILING IN THE

ADVANCING TRANSCRIPTION BUBBLE

mRNA (messenger RNA)• mRNA is synthasized from DNA by

enzyme RNA Polymerase.• Introns and Exons.(Hetrogenous hnRNA)• Most hetrogenous,highly elongated and

short lived.• Has 5’—3’ polarity complimentary to

coding strand.• For protein synthesis.• Stable in Euk. Unstable in Pro.

tRNA (Transfer RNA)• Much smaller in size (75-90

ribonucleotide)Clover leaf shaped.

• 15% of total cellular RNA.

• 20 species of tRNA.

• It acts as adaptor molecule.

• 40 different tRNA.

• Five arms of tRNA: 1:D-Arm; 2:TUC-Arm 3: CCA Arm(Acceptor) 4: Anticodon Arm 5: Variable Arm or Extra Arm (determine Sp.).

tRNA• Amino Acid acceptor arm 7 base pair

stem• 5’—3’ CCA.Links Amino Acid.AA are

linked co-valently to corresponding tRNA by enz.AAtRNA Synthetase.

• Anticodon Arm Has 5-bp loop and triplet nucleotide sequence(anticodon) complimentary to codon.

• DHU arm .3-4 base pairs.• TUC arm stem has 5 bp.• Extra arm 3—5bp.

RIBOSOMAL RNA• Are present in Ribosomes in association

with many polypeptides.• 80 % of total cellular RNA.• Very complex in Euc. Two sub-units

(60S)and (40S)• Protein Synthesis.• Larger unit has 3 RNA ie 5S,5.8S,28S

with 50 polypeptides.• Smaller subunit has 18S and 30

polypeptides.

MITOCHONDRIAL DNA

• 54 out of 67 genes are coded by nuclear genes,rest are coded by Mitochondria.(mt)

• It form 1% of total cellular DNA.

• It codes 13 proteins that play key role in the respiratory chain.

Features of mtDNA• Circular,double stranded• Contains 16,569 bp• Encodes 13 protein subunits.• Encodes 22 mt. tRNA molecules.• Encodes(16s) and a small (12s)mt rRNA• Very few untranslated sequences.• High mutation rate (5-10 times to nucl.)• M for M (Maternaly inherited Mitochondria)

Discovering the structure of DNA

• DNA = Deoxyribose nucleic acid

• Made out of sugars (deoxyribose), phosphates

and nitrogen bases

The mother of all biomolecules

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1ffk

Large subunit of the ribosome(proteins at least)

• Mitochondria

Function- ATP synthase, fatty acid oxidation, Krebs cycle, cytosolic Ca 2+ regulation.

Marker- Succinate dehydrogenase, glutamate dehydrogenase.

Redox potential of some redox system

Redox system Eo`(volt)

NADH/NAD+ -0.32

Succinate/Fumarate -0.32

FADH2/FAD 0.0

Coenzyme Q.H2/CoQ 0.10

Cytochrome b (Fe2+/Fe 3+) 0.12

Cytochrome c1 (Fe2+/Fe 3+) 0.22

Cytochrome a (Fe2+/Fe 3+) 0.29

Cytochrome a3 (Fe2+/Fe 3+) 0.39

HO2/1/2O2 0.82

• Endoplasm reticulum (2 type RER & SER)

Function- Production of excretory proteins, glycosylation, drug metabolism.

Marker- Glucose-6-phosphatase.

• Golgi complex

Function- Synthesis of lipoprotein, formation of lysosomes, secretion of proteins.

Marker- Galactosyle transferase, Fucosyle transferase.

• Peroxisomes

Function- Fatty acid oxidation, detoxification of H2O2.

Marker- Catalase, uric acid oxidase.

• Lysosomes

Function- Intracellular digestion and detoxification.

Marker- Acid phosphatase.