Molecular Biology Background

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Molecular Biology Background
Debasis Mitra Florida Tech
Credit Pevezner text-site
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Section1 What is Life made of?
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2 types of cells Prokaryotes v.s.Eukaryotes
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Life begins with Cell

• A cell is a smallest structural unit of an
  organism that is capable of independent
  functioning
• All cells have some common features

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Prokaryotes and Eukaryotes

• According to the most recent evidence, there are
  three main branches to the tree of life.
• Prokaryotes include Archaea (ancient ones) and
  bacteria.
• Eukaryotes are kingdom Eukarya and includes
  plants, animals, fungi and certain algae.

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Prokaryotes and Eukaryotes, continued
Prokaryotes Eukaryotes
Single cell Single or multi cell
No nucleus Nucleus
No organelles Organelles
One piece of circular DNA Chromosomes
No mRNA post transcriptional modification Exons/Introns splicing
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Overview of organizations of life

• Nucleus library
• Chromosomes bookshelves
• Genes books
• Almost every cell in an organism contains the
  same libraries and the same sets of books.
• Books represent all the information (DNA) that
  every cell in the body needs so it can grow and
  carry out its vaious functions.

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Chromosomes

• Organism Number of base pair
  number of Chromosomes
• --------------------------------------------------
  --------------------------------------------------
  -----
• Prokayotic
• Escherichia coli (bacterium) 4x106 1
• Eukaryotic
• Saccharomyces cerevisiae (yeast) 1.35x107 17
• Drosophila melanogaster(insect) 1.65x108 4
• Homo sapiens(human) 2.9x109 23
• Zea mays(corn) 5.0x109 10

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Bio-molecules

• Nucleic acids (DNA, RNA) Library of life
• Proteins Workhorse of life
• Fatty acids, carbohydrates, and other supporting
  molecules

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DNA

• DNA has a double helix structure which composed
  of
• sugar molecule
• phosphate group
• and a base (A,C,G,T)
• DNA always reads from 5 end to 3 end for
  transcription replication
• 5 ATTTAGGCC 3
• 3 TAAATCCGG 5

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DNA, RNA, and the Flow of Information
Replication
Transcription
Translation
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Proteins Functions

• Structural
• Enzymes
• Information exchange (e.g., across cell walls)
• Transporting other molecules (e.g., oxygen to
  cells)
• Activating-deactivating genes
• Etc.

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Proteins

• Amino acids
• Protein is a chain of residues
• 20 to 5000 long, typically a few hundred long

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Protein structure

• Important for its function
• Primary structure sequence
• Secondary structure a few topological features
• Tertiary structure 3D folding
• Quaternary structure Protein complex

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Protein Folding

• Proteins tend to fold into the lowest free energy
  conformation.
• Proteins begin to fold while the peptide is still
  being translated.
• Proteins bury most of its hydrophobic residues in
  an interior core to form an a helix.
• Most proteins take the form of secondary
  structures a helices and ß sheets.
• Molecular chaperones, hsp60 and hsp 70, work with
  other proteins to help fold newly synthesized
  proteins.
• Much of the protein modifications and folding
  occurs in the endoplasmic reticulum and
  mitochondria.

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Protein Folding (contd)

• The structure that a protein adopts is vital to
  its chemistry
• Its structure determines which of its amino acids
  are exposed carry out the proteins function
• Its structure also determines what substrates it
  can react with

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Nucleic acids

• Two types
• DNA Deoxy-ribonucleic acid
• RNA Ribonucleic acid

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Nucleic acids

• Sugar molecule chain forms the base of the
  polymer
• Two types of sugar ribose (RNA), 2-deoxyribose
  (DNA)

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Nucleic acids DNA

• 4 types of bases connected to sugar molecules
  Adenine (a), Guanine (g), Thymine (t) and
  Cytosine (c)
• A and T forms strong bonds, and so do G and C

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• The Purines

• The Pyrimidines

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DNA

• DNA has a double helix structure which composed
  of
• sugar molecule
• phosphate group
• and a base (A,C,G,T)
• DNA always reads from 5 end to 3 end for
  transcription replication
• 5 ATTTAGGCC 3
• 3 TAAATCCGG 5

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Nucleic acids DNA

• Double stranded two strands of sugar
  molecule-chains
• Each strand is directed 5 to 3
• Attached inside by base-pairings (a-t and g-c)

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Double helix of DNA

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Discovery of DNA

• DNA Sequences
• Chargaff and Vischer, 1949
• DNA consisting of A, T, G, C
• Adenine, Guanine, Cytosine, Thymine
• Chargaff Rule
• Noticing A?T and G?C
• A strange but possibly meaningless phenomenon.
• Wow!! A Double Helix
• Watson and Crick, Nature, April 25, 1953
• Rich, 1973
• Structural biologist at MIT.
• DNAs structure in atomic resolution.

Crick Watson
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Nucleic acids DNA

• Each strand is complementary and reverse to the
  other
• If sagacgt
• reverse(s)tgcaga
• reverse-complement(s)acgtct
• Double-strand 5--agacgt-gt3
• 3lt-t ctgca5

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Nucleic acids DNA

• 3D structure is helical
• Double-stranded helix like step ladder
• Each unit is a base pair (sugar-base-base-sugar)
• DNAs in cells are chromosomes (human chromosome
  3(109) bp long)
• Squeezed 3D structure in cell may have functional
  importance not well studied

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DNA Replication

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Nucleic acids RNA

• Replace t with u (uracil) as base
• May or may not be (mostly not) double stranded
• Functions Information storage like DNA,
  sometimes workhorse like proteins
• Possible evolutionary precursor to DNA and protein

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Genetic code

• Proteins do almost all the works!!
• Information for coding proteins are stored on
  DNAs (or RNAs) genes
• Three consecutive bases on a gene codes an amino
  acid, or the STOP code codon
• The table is called genetic code

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Cell Information Instruction book of Life

• DNA, RNA, and Proteins are examples of strings
  written in either the four-letter nucleotide of
  DNA and RNA (A C G T/U)
• or the twenty-letter amino acid of proteins. Each
  amino acid is coded by 3 nucleotides called
  codon. (Leu, Arg, Met, etc.)

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Overview of DNA to RNA to Protein

• A gene is expressed in two steps
• Transcription RNA synthesis
• Translation Protein synthesis

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Central Dogma of Biology

• The information for making proteins is stored
  in DNA. There is a process (transcription and
  translation) by which DNA is converted to
  protein. By understanding this process and how
  it is regulated we can make predictions and
  models of cells.

Assembly
Protein Sequence Analysis
Sequence analysis
Gene Finding
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Transcription

• Genes are transcribed to proteins typically one
  gene to one protein
• Genes are subsequenes on chromosomes started by a
  promoter region, ended around a stop codon

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Transcription

• Steps
• DNA is split over gene after promoter is
  recognized (may have other regulatory regions
  upstream)
• mRNA is copied from the gene
• Exons are spliced out from the mRNA keeping the
  introns only
• Ribosome (rRNA and protein complex) works on mRNA

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Transcription

• The process of making RNA from DNA
• Catalyzed by transcriptase enzyme
• Needs a promoter region to begin transcription.
• 50 base pairs/second in bacteria, but multiple
  transcriptions can occur simultaneously

http//ghs.gresham.k12.or.us/science/ps/sci/ibbio/
chem/nucleic/chpt15/transcription.gif
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Definition of a Gene

• Regulatory regions up to 50 kb upstream of 1
  site
• Exons protein coding and untranslated regions
  (UTR)
• 1 to 178 exons per gene (mean 8.8)
• 8 bp to 17 kb per exon (mean 145 bp)
• Introns splice acceptor and donor sites, junk
  DNA
• average 1 kb 50 kb per intron
• Gene size Largest 2.4 Mb (Dystrophin). Mean
  27 kb.

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Translation

• tRNA are attached to codons on mRNA
• On the other end the tRNA attracts appropriate
  amino acid
• Amino acids are zipped up
• No tRNA for STOP codon
• Every step is facilitated by appropriate enzyme
• Central Dogma of biology

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Translation, continued

• Catalyzed by Ribosome
• Using two different sites, the Ribosome
  continually binds tRNA, joins the amino acids
  together and moves to the next location along the
  mRNA
• 10 codons/second, but multiple translations can
  occur simultaneously

http//wong.scripps.edu/PIX/ribosome.jpg
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Revisiting the Central Dogma

• In going from DNA to proteins, there is an
  intermediate step where mRNA is made from DNA,
  which then makes protein
• This known as The Central Dogma
• Why the intermediate step?
• DNA is kept in the nucleus, while protein
  sythesis happens in the cytoplasm, with the help
  of ribosomes

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The Central Dogma (contd)
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Open Reading Frame

• Three reading frames in a strand
• Complementary strand may have another three frames

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Types of chromosomes

• Procaryotes (bacteria, blue algae) circular
• Eucaryotes (has nuclear wall) diploid (human has
  23 pairs)
• Homologous genes and alleles (e.g., human
  hemoglobin of type A, B, and O)
• Haploid chromosomes in Eucaryote sex cells

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DNA Sequencing

• A DNA fragment is split at each position starting
  from one end
• Four tubes one containing molecules ending with
  G, one with A, one with T and another one with C
• Electrophoresis separates each chunk of different
  size in each tube page 22
• Information is recombined to sequence the DNA
  chunk
• Can be done for the size of only 1K bp long chunk

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DNA Sequencing

• Human DNA is 109 bp long
• Restriction enzyme cuts at restriction sites (a
  product of genetic engineering) page 18
• After sequencing, information from fragments need
  to be recombined to get the broader picture

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DNA Sequencing

• Depends on finding restriction site/enzyme for
  fragmenting DNA of appropriate size
• Privately funded Tiger project (Celera now) used
  heat and vibration to create fragments
• Recombining information is no longer trivial
  because fragments location is no longer known
• Needed Fragment assembly algorithm

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DNA Sequencing

• Needs multiple copies of DNAs
• Recombinant DNA by biologically copying them
  within host organisms
• Polymerase Chain Reaction heat and tear two
  strands of DNA, then let each strand attract
  nucleic acids to form double stranded DNA, repeat