DNA consists of 4 bases DNA composition

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DNA and RNA Molecules of heredity

• DNA consists of 4 bases (DNA composition)
• Genes are made from DNA The Avery-MacLeod-McCarty
  and the Hershey-Chase experiments
• The discovery of DNA double helix
• DNA replication is semiconservative
• Melting temperature
• Length of DNA
• RNA and single stranded DNA viruses

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DNA consists of 4 kinds of bases

• Bases are-----gt Purines and pyrimidines
• Purines are A and G
• Pyrimidines are T and C
• Both DNA are RNA contain pyrimidine C but they
  differ in their second pyrimidine
• DNA-----gt T
• RNA-----gt U
• Sugar in DNA is deoxyribose
• Sugar in RNA is ribose

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DNA and RNA

• Nucleotide basesugarPhosphate
• Nucleosidebasesugar
• Note that the atoms in the rings of the bases are
  numbered 1-6 in prymidines and 1-9 in purines,
  while the carbons in the pentose are numbered
  1-5, thus when you refer to 5 carbon, you are
  specifying a C in pentose rather an atom in the
  base.
• In prymidines N-1 is bound to C-1 carbon in
  sugar, in purines N-9 to C-1.

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Numbering purines
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Structure of DNA

• DNA is a polynucleotide that contains many
  nucleotides covalently linked to each other by
  3,5-phoshodiester bonds
• 5OH-P-3OH
• The resulting long chain has polarity
• 5-------?3
• Phosphodiester bond can be cleaved by
• Chemicals
• Enzymes
• Deoxyribonuclease, ribonuclease, endonuclease,
  exonuclease, restriction endonucleases

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Summary

• Genetic information flows
• DNA--?RNA---? Proteins
• Purines and pyrimidines are building blocks of
  RNA and DNA
• ImportantPyrimidines look something like purines
  except that they are not so pure any more, having
  been CUT (Cytosine, Uracil, Thymine) in half,
  leaving a single hexagonal ring rather than the
  combined pure hexagonal-pentagonal structure of
  the purines. Thus purines are pure and unCUT

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Length of DNA

• DNA must comprise many nucleotides to carry the
  genetic information necessary for evn the
  simplest organisms.
• E. Coli genome is a single DNA molecule
  consisting of two chains of 4.6 million
  nucleotides.

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DNA molecules from higher organisms can be larger

• The human genome has 3 billion nucleotides (22
  autosomes, x and y sex chromosomes)
• Largest DNA is found in the Indian muntjak, as
  Asiatic deer, it is as large as human genome but
  it is distributed in only 3 chromosomes (each
  contain 1 billion nucleotides)!

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The d-helix is stabilized by H-bonds and
hydrophobic interactions

• Watson-Crick model of DNA
• Two helical chains are coiled around a common
  axis. The chains run in opposite directions.
• The sugar-P backbone is on the outside, the bases
  are on the inside.
• The bases are perpendicular to the helix axis,
  the adjacent bases are separated by 3.4 A, the
  helical structure repeats every 34 A, so, there
  are 10 bases /turn.
• The diameter of the helix is 20A.
• How is such a regular structure able to
  accommodate an arbitrary sequence of bases?

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Information is transferred by DNA not proteins

• Avery-MacLeod-McCarthy In 1944, 3 scientists
  made the discovery that the substance active in
  transforming Type R bacteria to virulance was in
  fact DNA!
• Hershey-Chase In 1952, Alfred Hershey and Martha
  Chase did an elegant experiment to trace the
  fates of 2 major components of bacteriophage-coat
  protein and DNA- following injection. They took
  advantage of the fact that DNA-? lack S
• Proteins-----? lack P

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Density-gradient equilibrium sedimentation

• Parent DNA is labeled with 15N by growing E. Coli
  in 15N containing medium (15NH4Cl)
• Then transfer E. Coli in 14N containing medium.
• Look at the distribution.
• If replication is semiconservative, DNA molecules
  isolated from cells after one round should
  contain 50/50 mixture of 15N 14N (intermediate
  density)

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Semiconservative replication of DNA proven by
density gradient replication
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Nucleic acid chemistry

• DNA and RNA can be denatured
• Denaturation Complete unwinding
• As temperature increases, more H-bonds are
  broken.
• Melting temperature (Tm) the T where number of
  dublex DNA is equal to the number of separated
  DNA( or half of the helical structure of DNA is
  lost)
• UV is used to measure the extent of denaturation
  (260 nm)
• Absorbance at 260 nm increases when double helix
  is melted Hyperchromism

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Tm melting temperature
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Many DNA molecules are circular and supercoiled

• EM shows that most DNA molecules are circular.
• Circular---? continuity of the DNA chain!
• Not all DNA are circular but some linear.
• Some interconvert between linear and circular.
• Supercoiled DNA and relaxed DNA
• DNA twisted counterclockwise--? supercoil
• DNA twisted clockwise----? - supercoil
• DNA with no supercoils-----?relaxed

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Negative and positive coiling
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Linking number

• The number of times that one strand of a dublex
  DNA molecule winds around the other is the
  linking number for that DNA.
• The linking number for a relaxed B-DNA molecule
  is
• the number of base pairs that has/ 10.4
• 10.4 is the number of base pairs per turn

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Supercoiling
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Linking number

• 260 bp DNA (B-DNA)?Lk?
• If you unwound this DNA by 2 turns (Lk?)
• Circular DNA molecules that have the same
  nucleotide sequence but different linking numbers
  are called TOPOISOMERS.
• Why is supercoiling biologically important?
• A supercoiled DNA has a more compact shape
  (packaging becomes easy)
• Supercoiling affects DNAs interactions with
  other molecules

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The genes of some viruses are made of RNA

• Genes in all pro and eukaryotes are made of DNA.
• In viruses, genes are made of either DNA or RNA
• RNA is like DNA but
• Sugar is ribose
• There is U instead T
• RNA can be single or double stranded. RNA cannot
  form a double helix of B-DNA type because of
  steric interference by the 2-OH groups of its
  ribose units. The 2-OH of ribose prevents RNA
  forming a classic Watson-Crick B helix.Instead it
  forms A-DNA. It is rather tilted DNA.

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All the genetic info of the virus is contained in
its RNA

• Tobacco mosaic virus infects tobacco plants.
• It is a single stranded virus and surrounded by
  protein units.
• RNA tumor viruses and other retroviruses
  replicate via ds-DNA intermediates.
• RNA viruses produce malignant tumors.
• Raus sarcoma virus is one of them.
• RNA tumor viruses replicate through DNA
  intermediates, HIV-1 is one of them. They are
  known as retroviruses because information flows
  BACKWORD not from
• DNA---? RNA but RNA---?DNA

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Infection of Tobacco Hybrid mosaic virus
particles
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Single-stranded nucleic acids can adopt eloborate
structures

• SS-nucleic acids often fold back on themselves to
  form well-defined structures.
• Ribosomes have these stuructures.
• The simplest and most common structural motif
  formed is a stem-loop, created when two
  complementary sequences within a single strand
  come together to form double-helical structures.
• Sometimes there is mismatched and unmatched bases
  (Fig 5.20).

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DNA replication

• DNA polymerase catalyzes phosphodiester bond
  formation, template directed enzyme.
• The strand is synthesized in the 5---gt3
  direction
• (DNA)n dNTP----gt (DNA)n1 Ppi
• Primer is needed
• All four activated precursors needed
• Nucleophilic attack by the 3-OH group of the
  primer on the innermost P atom of the dNTP.

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Chain Elongation
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Several kinds of RNA play key roles in gene
expression
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Several kinds of RNA

• mRNA
• Template for protein synthesis (translation)
• mRNA is produced for each gene
• Average length of an mRNA in prokaryotes is about
  1.2 kb.
• tRNA
• Carries amino acids in an activated form to the
  ribosome for peptide-bond formation.
• There is at least one tRNA for each aa
• It has 75 nucleotides
• rRNA
• Major component of ribosomes (23S, 16S, 5S)
• Most abundant of all.

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Eukaryotic cells have additional small RNA
molecules

• snRNA (small nuclear RNA)
• Splicing of RNA exons
• A small RNA
• Is essential component of Signal-recognition
  particle
• MicroRNA (miRNA)
• Small noncoding RNA that bind to complementary
  RNA and inhibit their translation
• Small interfering RNA (SiRNA)
• Bind to mRNA and facilitate their degradation
• RNA
• Component of telomerase

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RNA polymerase, active site has magnesium
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RNA poly requires

• 1. A template
• dsDNA (preferred), RNA is not effective!
• 2. Activated precursors
• All 4 ribonucleoside triphosphates-ATP, GTP,
  UTP,and CTP
• 3. A divalent metal ion

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Synthesis of RNA is like DNA

• Direction is the same
• The mechanism is the same
• Driven forward by the hydrolysis of PPi
• In contrast with DNA poly, RNA poly DOES NOT
  require a primer!
• Does not proofread!

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RNA poly take instructions from DNA template
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evidences

• Base composition of newly synthesized RNA
• Hybridization experiments

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Transcription begins near promoter sites
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Transcription begins near promoters sites
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1. RNA poly proceeds along The DNA
template Until it makes a terminator
Sequence! Termination signal Base-paired
hairpin! (rich in GC) followed by polyU 2. or
by Rho protein..
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Modification of mRNA
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tRNA adaptors
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Genetic code

• 1. 3 nucleotides encode for an aa.
• 2. the code is nonoverlapping
• 3. no punctiotions
• 4. genetic code is degenerate
• Some aas are encoded by more than one codon, so
  degenerate.
• The number of codons for a particular aa
  correlates well with its frequency of occurance
  in proteins!

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More info

• Synonym Codons that specify the same aa. (CAU
  and CAC are synonyms)
• Most synonyms differ only in the last base of the
  triplet
• XYC XYU always encode for the same aa.

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What is the biological significance of the
extensive degeneracy of the genetic code?

• If the code were NOT degenerate, 20 codons would
  code for 20 aa and 44 would lead to chain
  termination! The probability of mutating chain
  termination would therefore be much higher with a
  nondegenerative code.
• Chain termination mutations -? inactive proteins
• Substitutions of one aa for another are usually
  harmless
• So, degeneracy decreases harmful effects of
  mutations!

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Pp chains in bacteria starts with A modified aa,
formylMet Initiator tRNA has fMet This recognizes
AUG!!!
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In eukaryotes, the AUG closest to the 5end of
mRNA is the start signal!
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The genetic code is nearly universal The genetic
code of mitochondria is different because mtDNA
encodes A distinct set of tRNAs.
At least 16 organisms deviate from the standard
genetic code. Thus, genetic code is nearly but
NOT absolutely universal!
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Most eukaryotic genes are mosaics of introns and
exons
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Introns and exons

• In 1977, genes were found to be discontinues in
  higher organisms.
• Definitions
• Consensus sequences the base sequences that are
  almost identical (there may be one or two base
  differences).
• Intervening sequences not copied, not expressed,
  introns
• Do bacteria have introns?
• Exon Expressed gene, the ones that are retained
  in mature RNA are called exons!

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Prove for introns

• mRNA is hybridized to a ssDNA.
• If the gene is continues (no introns), a single
  loop will be seen (Figure A)
• Two loops of ssDNA and a loop of dsDNA are seen
  if the gene contains an intervening sequence!
• Intervening sequences are not used to make mRNA
  (Figure B)

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Detection of introns by EM
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RNA processing generates mature RNA
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Consensus sequence for the splicing of mRNA
precursors Spliceosomes do the splicing (proteins
and small RNA molecules)
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Exon shuffling

• Many exons encode discrete structural and
  functional units of proteins.
• Hypothesis states that new proteins are made in
  evolution by rearangement of exons by a process
  called exon shuffling.
• Exon shuffling is a rapid and efficient means of
  generating novel genes.

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Exon shuffling