Fundamentals of Nucleic Acid Biochemistry: DNA

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Fundamentals of Nucleic Acid Biochemistry DNA

• Donna C. Sullivan, PhD
• Division of Infectious Diseases
• University of Mississippi Medical Center

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The Central Dogma ofMolecular Biology
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THE GENOME
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THE GENETIC MATERIAL
Is arranged as
Is usually
Can be
Genes
RNA
DNA
Gives rise to
Two aspects
Arranged as
Which is of three types
Which produces
Gene expression
Gene structure
Double helix
Ribosomal (rRNA)
Messenger (mRNA)
Transfer (tRNA)
specifies
Composed of
specifies
Component of
In prokaryotes
requires
In eukaryotes
Anticodons and amino acids
Ribosomes
Codons
Relatively simple
Transcription
Complex
by
Translation
Sugar-P backbone
Nitrogenous bases

Genes have
Genes grouped in
RNA polymerase
Which are involved in
Known as
Introns Exons 5 cap 3 poly(A)
Purines
Pyrimidines
Which produces
Operons
Proteins
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STRUCTURE OF NUCLEIC ACIDS

• Primary structure
• Polymers of nucleotides
• Nucleotides
• Phosphate group
• Sugar
• Base

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TWO TYPES OF SUGAR IN NUCLEIC ACIDS
Both retain a 3 hydroxyl group
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Purines Pyrimidines Diagrams
Source http//www.mun.ca/biology/scarr/2250_DNA_b
iochemistry.htm
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Structure of Nucleotides
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DNA AND RNA BASIC STRUCTURE
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Repeating Nucleotide Subunits In DNA and RNA
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Functions of DNA

• DNA serves two important functions contributing
  to cellular homeostasis
• Stable storage of genetic information
• Transmission of genetic information
• DNA provides the source of information for the
  synthesis of all the proteins in a cell, and it
  serves as a template for the faithful replication
  of genetic information that is ultimately passed
  into daughter cells.

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STRUCTURE OF DNA

• SUGAR
• Deoxyribose
• Phosphate group
• Nitrogen containing base
• Adenine
• Guanine
• Cytosine
• Thymidine

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Polymerized Nucleotides
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Directionality 5 to 3
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DOUBLE HELIX OF DNA
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CONFORMATIONS OF DNA
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THE DOUBLE HELIX FORMS

• B form (Major form)
• Right handed
• Two helical grooves which allow protein binding
• A form
• Found in RNA-DNA or RNA-RNA helices
• More compact than B form
• Z form (Zig-Zag)
• Left handed
• Composed of alternating G and C residues

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GROOVES OF DNA
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DNA MOLECULAR CONFORMATIONS

• Linear ds DNA found in eukaryotic cell
• Other conformations found in
• Prokaryotic cells Circular ds DNA
• Viruses
• Circular ds DNA (Adenoviruses, SV40)
• Linear ss DNA (Parvoviruses)
• Circular partially ds DNA (HBV)

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DNA SYNTHESIS

• Semiconservative
• New DNA contains one old strand plus one
  new strand
• Bidirectional
• Each of the old strands is copied
• Each strand is copied simultaneously
• Initiates at a common site origin of replication

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DNA REPLICATION
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DNA REPLICATIONSemi-conservative
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DNA REPLICATION ORIGIN

• Replication of DNA begins at specific sites
• E. coli ori (1) 240 bp
• Yeast ori 400 on 17 chromosomes
• Contain multiple short repeated sequences
• Repeat units recognized by multimeric origin
  binding proteins
• Usually contain an AT rich region

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DNA POLYMERASES

• Prokaryotic DNA Polymerases
• DNA pol I
• DNA pol II
• DNA pol III
• Eukaryotic DNA Polymerases
• DNA pol ?
• DNA pol ?
• DNA pol ? (Two additional DNA pols ? and ?)

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CHARACTERISTICS OF DNA POLYMERASES

• Some DNA Polymerases have proof-reading function
• Can not separate (uncoil or unwind) DNA
• Must have a primer to initiate replication,
  primers synthesized by RNA Polymerases
• Catalyze nucleotide addition at the 3 OH,
  strands only grow in 5 to 3 direction

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DNA REPLICATION5 3 DIRECTION OF SYNTHESIS
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OTHER ENZYMES AT THE INTITIATION SITE

• DNA gyrase unwinds supercoils
• DNA helicase separates double helix
• Single stranded DNA binding protein (ss DBP)
• Primase a type of RNA Polymerase
• Topoisomerase

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UNWINDING SUPERCOILS

• Supercoiling occurs when
• Two ends of DNA are fixed (ie, circular)
• DNA is in long helical structure (ie, large
  chromosome)
• Supercoiling occur during
• Replication of DNA
• Transcription of RNA from DNA
• Binding by some types of protein
• Supercoiling is relieved by helicases,
  topoisomerases

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LEADING AND LAGGING STRANDS OF DNA

• Leading strand of daughter DNA
• Synthesis occurs continuously from a single RNA
  primer at its 5 end
• Lagging strand of daughter DNA
• Synthesis occurs discontinuously from multiple
  RNA primers that are formed on parental strand as
  each new region of DNA is exposed at the growing
  fork
• Okazaki fragments
• Joined by DNA ligase to form continuous DNA

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Termination

• Once the new strands are complete, the molecules
  rewind automatically in order to regain their
  stable helical structure.

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Termination

• A problem is created once the RNA primer is
  removed from the 5 end of each daughter strand,
  there is no adjacent fragment for which new
  nucleotides can be added to fill this gap,
  resulting in a slightly shorter daughter
  chromosomes.
• This occurrence is not a problem in circular DNA,
  but human cells loose about 100 base pairs from
  each end of each chromosome with each replication

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Termination

• This loss of genetic material could result in
  critical code being eliminated, however there are
  buffer zones of repetitive nucleotide sequences,
  called the telomeres.
• In humans the sequence is TTAGGG repeated several
  thousand times.

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Telomeres and Cell Death

• Their erosion does not affect cell function, but
  protects against lost of important genetic
  material.
• The erosion of the telomeres are related to the
  death of the cell.

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Termination

• Thus, extension of telomeres is linked to longer
  lifespan for the cell.
• Enzyme telomerase responsible for extension.
• Gene that codes for telomerase is directly linked
  to the longevity in worms and fruit flies
• Cancer cells also contains telomerase.

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Proofreading and Correction

• Errors occur in DNA replication fairly
  frequently the wrong base gets inserted due to
  the peculiarities of nucleotide chemistry

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Proofreading and Correction

• DNA polymerase has editing function that removes
  most of the incorrect bases.
• DNA pol detects the absence of hydrogen bonding
  (when a mismatch occurs), removes the incorrect
  base and inserts the correct one using the parent
  strand as a template.
• This complex process of replication is known as
  the replication machine.

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Nucleic Acid Modifying Enzymes

• Restriction endonucleases (molecular scalpels)
• DNA polymerases (synthesize DNA)
• DNA ligases (join DNA strands by forming a
  phosphodiester bond)
• Kinases (phosphorylation of 5-terminus of DNA
  molecule)

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Nucleic Acid Modifying Enzymes

• Phosphatases (dephosphorylate 5-terminus of DNA
  molecule)
• Ribonucleases (digest RNA molecule. Example
  RNase A)
• Deoxyribonucleases (digest DNA molecules)

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Restriction Endonucleases (RE)

• Found only in microorganisms
• Exhibit novel DNA sequence specificities
• gt2000 distinct restriction enzymes have been
  identified
• Function as homodimer recognize symmetrical
  dsDNA (palindromes)
• Utilized in the digestion of DNA molecules for
  hybridization procedures or in the direct
  identification of mutations

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Restriction Enzymes Recognize Palindromes

• Palindrome reads the same in both directions
• BOB
• Able was I ere I saw Elba. (Napoleon Bonapart,
  following his exile from the European continent
  to the island of Elba)
• Sequences directly opposite one another on
  opposite strands of the ds DNA molecule

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Restriction Endonucleases

• Recognize specific sequences of 4, 5, or 6
  nucleotides
• Cut by breaking the phosphodiester bond in both
  strands
• Cutting genomic DNA with a RE results in many
  fragments of different sizes
• The smaller the recognition sequence the larger
  the number of fragments produced

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Restriction Enzymes
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Restriction Enzymes
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Typical Restriction Digestion Reaction

• Reactions are composed of DNA template,
  restriction enzyme, 10X buffer, and distilled
  water.
• The required amounts of these components can be
  calculated based on the number of specimens to be
  digested
• ?L DNA (10 ?g/rxn)
• ?L restriction enzyme
• ?L 10X buffer
• ?L distilled water

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Modifying Enzymes

• DNA ligase
• catalyses formation of bonds between 5-P and
  3-OH groups on backbone of DNA
• ligate blunt end or sticky ends
• can repair nicks in DNA
• DNA polymerase
• require primers to extend and copy DNA
• all extend 5?3 by adding on to 3-OH
• make a reverse, complimentary copy

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Modifying Enzymes

• Nucleases (exonucleases)
• cut DNA in non-sequence specific manner
• can digest DNA from either 5?3 or 3?5
  direction
• prefer ssDNA
• proofreading function of polymerase
• Alkaline phosphatase
• removes 5 P, prevents recircularization of
  plasmids

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Modifying Enzymes

• DNAse
• non-specifically digests DNA, ds or ss
• commonly found on most surfaces, including hands
• RNAse
• many different types, may be specific for ssRNA
  or RNA/DNA hybrids (RNAse H)
• extremely common (especially on hands), very
  stable

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Replication of Chromosomes Mitosis
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Assortment of Chromosomes Meiosis
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Chromosomal crossover

• Refers to the process by which two chromosomes,
  paired up during prophase 1 of meiosis, exchange
  some portion of their DNA.
• Usually occurs when matching regions on matching
  chromosomes break and then reconnect to the other
  chromosome.
• The result of this process is an exchange of
  genes, called genetic recombination.

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Recombination of Genetic Material Crossing Over
Events
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Crossing Over Allelic Recombination
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GENE TRANSFER IN BACTERIA

• Transformation
• Binding and uptake of naked extracellular DNA by
  a competent, living bacterial cell
• Conjugation
• Transfer of DNA directly from one living
  bacterial cell to another
• Transduction
• Transfer of bacterial genes via a bacterial virus
  (phage) vector

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GENE TRANSFER IN BACTERIA

• Requires stabilization of new genes to avoid
  rapid degradation of imported linear DNA
  (covalently closed DNA--plasmids--survive)
• Homologous recombination
• Exchange of two nearly identical pieces of DNA
• Requires a DNA binding protein (DBP) called recA

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BACTERIAL TRANSFORMATION
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BACTERIAL CONJUGATION PLASMID DNA
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BACTERIAL CONJUGATION PLASMID DNA
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BACTERIAL CONJUGATION PLASMID DNA
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BACTERIAL CONJUGATIONPLASMID DNA
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BACTERIAL CONJUGATION CHROMOSOMAL DNA
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BACTERIAL CONJUGATION CHROMOSOMAL DNA
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Summary

• DNA is a polymer of nucleotides, storing genetic
  information in the order of the nucleotide
  sequence.
• DNA replication conserves the DNA sequence.
• The two strands of double-stranded DNA are
  antiparallel and complementary DNA can be
  manipulated in vitro using DNA-metabolizing
  enzymes.
• Recombination is a natural process in eukaryotes
  and prokaryotes to produce offspring with new
  genetic combinations (recombinants).
• Restriction endonucleases, ligase, and plasmids
  are used to make new genetic combinations in
  vitro.

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Fundamentals of Nucleic Acid Biochemistry RNA
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STRUCTURE OF RNA

• SUGAR
• Ribose
• Phosphate group
• Nitrogen containing base
• Adenine
• Guanine
• Cytosine
• Uracil

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THE NUCLEOTIDE RNA
OH OP-O-5CH2
BASE OH O
4C 1C H
H H H
3C 2C
OH 0H

Adenine Guanine Cytosine Uracil
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The Forms Of RNA Not Just Another Helix

• Does not normally exist as a double helix,
  although it can under some conditions
• Can have secondary structure
• Hairpins pairing of bases within 5-10 nt
• Stem-loops pairing of bases separated by gt50 nt
• Can have tertiary structure
• Pseudoknot, cloverleaf

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RNA Structures
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Structure Of RNA
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Structure of tRNA
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Structure of rRNA
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Transcription

• Transcription is the enzyme-dependent process of
  generating RNA from DNA.
• The process is catalyzed by a DNA-dependent RNA
  polymerase enzyme.
• Only coding segments of DNA (genes) are
  transcribed.
• Types of genes include structural genes (encode
  protein), transfer RNA (tRNA), and ribosomal RNA
  (rRNA).

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RNA Transcription vs. DNA Replication

• RNA replication
• Requires no priming
• Has many more initiation sites
• Is slower (50100 b/sec vs. 1000 b/sec)
• Has lower fidelity
• Is more processive

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Transcription

• Transition from DNA to RNA
• Initiation Gene recognition
• RNA polymerase enzyme and DNA form a stable
  complex at the gene promoter.
• Promoter Specific DNA sequence that acts as a
  transcription start site.
• Synthesis of RNA Proceeds using DNA as a
  template.
• Only one strand (coding strand) is transcribed,
  the other strand has structural function.
• Transcription factors are proteins that function
  in combination to recognize and regulate
  transcription of different genes.
• Termination signal

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RNA POLYMERASES

• Cellular RNA polymerase
• RNA pol I transcribes rRNA genes
• RNA pol II transcribes protein encoding genes
• RNA pol III transcribes tRNA genes
• Viral RNA polymerases
• Reverse transcriptase of retroviruses
• RNA dependent RNA polymerases of negative
  stranded viruses

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RNA Pol Enzymes(DNA-dependent RNA Pol)

• RNA Polymerase I transcribes most rRNA genes (RNA
  component of ribosomes).
• RNA Polymerase II transcribes structural genes
  that encode protein.
• RNA Polymerase III transcribes tRNA genes (for
  transfer RNAs).
• RNA Polymerase IV is the mitochondrial RNA
  polymerase enzyme.

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TBP Transcription binding protein TF
Transcription Factor
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RNA TRANSCRIPTION
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General Organization of a Eukaryotic Gene
Promoter/Enhancer
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Gene Structure
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Nuclear Processing of RNA

• Chemical modification reactions (addition of the
  5 CAP)
• Splicing reactions (removal of intronic
  sequences)
• Polyadenylation (addition of the 3 polyA tail)

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Processing of mRNA

• In eukaryotes, the mRNA molecule that is released
  after transcription is called precursor mRNA or
  pre-mRNA.
• It undergoes several changes before being
  exported out of the nucleus as mRNA.

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Processing of mRNA

• 5 end is capped with a modified form of the G
  nucleotide known as the 5 cap
• At the 3 end, an enzyme adds a long series of A
  nucleotides referred to as a poly-A tail
• It serves to protect the mRNA from enzymes in the
  cytoplasm that may break it down
• The greater the length of the poly-a tail, the
  more stable the mRNA molecule

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RNA Transcription and Processing

• The process of RNA transcription results in the
  generation of a primary RNA transcript (hRNA)
  that contains both exons (coding segments) and
  introns (noncoding segments).
• The noncoding sequences must be removed from the
  primary RNA transcript during RNA processing to
  generate a mature mRNA transcript that can be
  properly translated into a protein product.

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RNA Processing

• Capping of the 5-end of mRNA is required for
  efficient translation of the transcript (special
  nucleotide structure).
• Polyadenylation at the 3-end of mRNA is thought
  to contribute to mRNA stability (PolyA tail).
• Once processed, the mature mRNA exits the nucleus
  and enters the cytoplasm where translation takes
  place.

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RNA ProcessingBiogenesis of Mature mRNA
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RNA ProcessingFundamentals of RNA Splicing
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RNA Processing Spliceosome

• Ribonucleoproteins (snRNPs) function in RNA
  processing to remove intronic sequences from the
  primary RNA transcript (intron splicing).
• Alternative splicing allows for the generation of
  different mRNAs from the same primary RNA
  transcript by cutting and joining the RNA strand
  at different locations.
• snRNPs are composed of small RNA molecules and
  several protein molecules.
• Five subunits form the functional spliceosome.

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RNA ProcessingAlternative Splicing of RNA
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Structural Genes Encode Proteins

• The majority of structural genes in the human
  genome are much larger than necessary to encode
  their protein product.
• Structural genes are composed of coding and
  noncoding segments of DNA.

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Structural Genes Encode Proteins

• The structure of a typical human gene includes
  informational sequences (coding segments termed
  exons) interrupted by noncoding segments of DNA
  (termed introns).
• The exon-introncontaining regions of genes are
  flanked by non transcribed segments of DNA that
  contribute to gene regulation.

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Factors That Control Gene Expression Include

• Cis elements are DNAsequences.
• Trans elementsare proteins.

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Control of Gene Expression

• Primary level of control is regulation of gene
  transcription activity.
• TATA box contained within the gene promoter
  provides binding sites for RNA polymerase.
• Enhancer sequences can be sited very far away
  from the gene promoter and provide for
  tissue-specific patterns of gene expression.

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Gene Enhancer Sequences

• Enhancer sequences are usually sited a long
  distance from the transcriptional start site.
• Enhancers maintain a tissue-specific or
  cell-specific level of gene expression.
• The gene promoter contains TATA box upstream of
  transcription start site.

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Protein Binding Sequences in the Promoter region
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Gene Regulation Two types of regulation

• Negative regulation
• Substrate induction (lac operon) gene OFF unless
  substrate is present
• End product repression (trp operon) gene OFF if
  end product is present
• Common in bacteria
• Positive regulation
• Gene is OFF until a protein turns it ON
• Regulatory proteins turn gene ON
• Occurs in eukaryotes

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Negative Regulation
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Positive Regulation
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Lac Operon

• Operon
• Gene organization in bacteria in which several
  proteins are coded by one mRNA
• Allows all proteins to be controlled together

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Differences Between Prokaryotes And Eukaryotes

• Prokaryote gene expression typically is regulated
  by an operon, the collection of controlling sites
  adjacent to protein-coding sequence.
• Eukaryotic genes also are regulated in units of
  protein-coding sequences and adjacent controlling
  sites, but operons are not known to occur.
• Eukaryotic gene regulation is more complex
  because eukaryotes possess a nucleus
  (transcription and translation are not coupled).

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Two Categories Of Eukaryotic Gene Regulation

• Short-term - genes are quickly turned on or off
  in response to the environment and demands of the
  cell.
• Long-term - genes for development and
  differentiation.

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Eukaryote Gene Expression Is Regulated At Six
Levels

• Transcription
• RNA processing
• mRNA transport
• mRNA translation
• mRNA degradation
• Protein degradation

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Transcription Control Of Gene Regulation
Controlled By Promoters

• Occur upstream of the transcription start site.
• Some determine where transcription begins (e.g.,
  TATA), whereas others determine if transcription
  begins.
• Promoters are activated by highly specialized
  transcription factor (TF) proteins (specific TFs
  bind specific promoters).
• One or many promoters (each with specific TF
  proteins) may occur for any given gene.
• Promoters may be positively or negatively
  regulated.

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Transcription Control Of Gene Regulation
Controlled By Enhancers

• Occur upstream or downstream of the transcription
  start site.
• Regulatory proteins bind specific enhancer
  sequences binding is determined by the DNA
  sequence.
• Loops may form in DNA bound to TFs and make
  contact with enhancer elements.
• Interactions of regulatory proteins determine if
  transcription is activated or repressed
  (positively or negatively regulated).

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Chromosome Structure, Eukaryote Chromosomes, And
Histones

• Prokaryotes lack histones and other structural
  proteins, so access to the DNA is
  straightforward.
• Eukaryotes possess histones, and histones repress
  transcription because they interfere with
  proteins that bind to DNA.
• Verified by DNAse I sensitivity experiments
• DNAse I readily degrades transcriptionally active
  DNA.
• Histones shield non-transcribed DNA from DNAse I,
  and DNA does not degrade as readily.

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Transcriptional State Of Eukaryotic Chromatin
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Chromosome Structure, Eukaryote Chromosomes, And
Histones

• If you experimentally add histones and promoter
  binding proteins histones competitively bind to
  promoters and inhibit transcription.
• Solution transcriptionally active genes possess
  looser chromosome structures than inactive genes.
• Histones are acetylated and phosphorylated,
  altering their ability to bind to DNA.
• Enhancer binding proteins competitively block
  histones if they are added experimentally with
  histones and promoter-binding TFs.
• RNA polymerase and TFs step-around the
  histones/nucleosome and transcription occurs.

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Transcription Factor Binding
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Genomic Imprinting(Silencing)

• Methylation of DNA inhibits transcription of some
  genes.
• Methylation usually occurs on cytosines or
  adenines.
• 5-methyl cytosine
• N-6 methyl adenine
• N-4 methyl cytosine
• CpG islands are sites of methylation in human
  DNA.
• CpG Island

ggaggagcgcgcggcggcggccagagaaaaa gccgcagcggcgcgcg
cgcacccggacagccgg cggaggcggg...
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DNA Methylation And Transcription Control

• Small percentages of newly synthesized DNAs (3
  in mammals) are chemically modified by
  methylation.
• Methylation occurs most often in symmetrical CG
  sequences.
• Transcriptionally active genes possess
  significantly lower levels of methylated DNA than
  inactive genes.
• A gene for methylation is essential for
  development in mice (turning off a gene also can
  be important).
• Methylation results in a human disease called
  fragile X syndrome FMR-1 gene is silenced by
  methylation.

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Hormone Regulation Short-term Regulation Of
Transcription

• Cells of higher eukaryotes are specialized and
  generally shielded from rapid changes in the
  external environment.
• Hormone signals are one mechanism for regulating
  transcription in response to demands of the
  environment.
• Hormones act as inducers produced by one cell and
  cause a physiological response in another cell.

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Hormone Regulation Short-term Regulation Of
Transcription

• Hormones act only on target cells with hormone
  specific receptors, and levels of hormones are
  maintained by feedback pathways.
• Hormones deliver signals in two different ways
• Steroid hormones pass through the cell membrane
  and bind cytoplasmic receptors, which together
  bind directly to DNA and regulate gene
  expression.
• Polypeptide hormones bind at the cell surface and
  activate transmembrane enzymes to produce second
  messengers (such as cAMP) that activate gene
  transcription.

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Examples Of Mammalian Steroid Hormones
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Hormone Regulation

• Genes regulated by steroid hormones possess
  binding regions in the sequence called steroid
  hormone response elements (HREs).
• HREs often occur in multiple copies in enhancer
  sequence regions.
• When steroid is absent Receptor is bound and
  guarded by chaperone proteins transcription
  does not occur.
• When steroid is present Steroid displaces the
  chaperone protein, binds the receptor, and binds
  the HRE sequence transcription begins.

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Model Of Glucocorticoid Steroid Hormone Regulation
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RNA Processing Control

• RNA processing regulates mRNA production from
  precursor RNAs.
• Two independent regulatory mechanisms occur
• Alternative polyadenylation where the polyA
  tail is added
• Alternative splicing which exons are spliced
• Alternative polyadenylation and splicing can
  occur together.
• Examples
• Human calcitonin (CALC) gene in thyroid and
  neuronal cells
• Sex determination in Drosophila

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Alternative Polyadenylation And Splicing Of The
Human CALC Gene In Thyroid And Neuronal Cells
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mRNA Transport Control

• Eukaryote mRNA transport is regulated.
• Some experiments show 1/2 of primary transcripts
  never leave the nucleus and are degraded.
• Mature mRNAs exit through the nuclear pores.

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mRNA Transport Control

• Unfertilized eggs are an example in which mRNAs
  (stored in the egg/no new mRNA synthesis) show
  increased translation after fertilization).
• Stored mRNAs are protected by proteins that
  inhibit translation.
• Poly(A) tails promote translation.
• Stored mRNAs usually have short poly(A) tails
  (15-90 As vs 100-300 As).
• Specific mRNAs are marked for deadenylation
  (tail-chopping) prior to storage by AU-rich
  sequences in 3-UTR.
• Activation occurs when an enzyme recognizes
  AU-rich element and adds 150 As to create a full
  length poly(A) tail.

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mRNA Degradation Control

• All RNAs in the cytoplasm are subject to
  degradation.
• tRNAs and rRNAs usually are very stable mRNAs
  vary considerably (minutes to months).
• Stability may change in response to regulatory
  signals and is thought to be a major regulatory
  control point.
• Various sequences and processes affect mRNA
  half-life
• AU-rich elements
• Secondary structure
• Deadenylation enzymes remove As from poly(A) tail
• 5 de-capping
• Internal cleavage of mRNA and fragment degradation

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Post-translational Control - Protein Degradation

• Proteins can be short-lived (e.g., steroid
  receptors) or long-lived (e.g., lens proteins in
  your eyes).
• Protein degradation in eukaryotes requires a
  protein co-factor called ubiquitin.
• Ubiquitin binds to proteins and identifies them
  for degradation by proteolytic enzymes.
• Amino acid at the N-terminus is correlated with
  protein stability and determines rate of
  ubiquitin binding.
• Arg, Lys, Phe, Leu, Trp 1/2 life 3 min
• Cys, Ala, Ser, Thr, Gly, Val, Pro, Met 1/2 life
  20 hrs

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Epigenetics

• The study of mechanisms by which genes bring
  about their phenotypic effects
• Epigenetic changes influence Phenotype without
  altering Genotype
• Changes in properties of a cell that are
  inherited but dont represent a change in genetic
  information.

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Epigenetics

• Non-sequence specific, heritable traits
• Transcriptional gene silencing (TGS)
• Imprinting
• X-inactivation
• RNA-induced transcriptional silencing (RITS)
• Post-transcriptional gene silencing (PTGS)
• RNA-induced silencing complex (RISC)
• G quartets
• Post-translational protein-protein interactions

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Angelman syndrome vs Prader-Willi syndrome

• Region of chromosome 15, most commonly by
  deletion of a segment of that chromosome.
• Maternal and paternal contribution express
  certain genes very differently due to sex-related
  epigenetic imprinting (biochemical mechanism is
  DNA methylation)
• In a normal individual, the maternal allele is
  methylated and the paternal allele is
  unmethylated.
• If the maternal contribution is lost or mutated,
  the result is Angelman syndrome.
• When the paternal contribution is lost, by
  similar mechanisms, the result is Prader-Willi
  syndrome

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RNAi

• First described in C. elegans
• Injecting antisense RNA into oocytes (ssRNA that
  is complementary to mRNA)
• Silences gene expression
• Also injected dsRNA into oocytes found it was 10X
  more potent in inhibiting expression
• Term RNAi

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DNA Associates With Histone Proteins To Form
Chromatin
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131
A Histone Code?

• Modifications of histones (usually the amino
  terminal tails) convey epigenetic information
• Types of modifications
• Acetylation and methylation of lysine.
• Methylation of arginine
• Phosphorylation of serine
• Ubiquitination of lysine.
• The histone code hypothesis posits that serial
  modifications provide a blueprint for reading
  chromatin -for transcription, replication,
  repair, and recombination.

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Remodeling Nucleosomes

• Changing the way nucleosomes bind to the DNA in
  chromosomes is important to allow access to the
  underlying DNA sequences during DNA replication,
  repair, recombination, and transcription.
• This occurs in three general ways
• Modification of the lys and Arg residues on the
  histone tails decreases the grip of the
  nucleosome on DNA and causes the nucleosome to
  slide more easily.
• Variant histones are added to pre-existing
  nucleosomes
• ATP-dependent protein remodeling" complexes
  cause nucleosomes to dissociate and/or slide
  along the DNA.

133
RNAi

• Higher eukaryotes produce a class of small RNAs
  that mediate silencing of some genes
• Small RNAs interact with mRNA in the 3UTR and
  this results in either mRNA degradation or
  translation inhibition
• Controls developmental timing in at least some
  organisms
• Used as a mechanism to protect against invading
  RNA viruses (plants)
• Controls the activity of transposons
• Formation of heterochromatin

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Mechanism of RNA Interference
135
Protein Synthesis and Function Chapter 3
136
Central Dogma
Central Dogma of the transfer of
biological information. DNA RNA protein
Nucleic acid sequence must be translated into an
amino acid sequence.
137
PROTEIN SYNTHESIS
138
Protein Translation
139
Initiation of Translation (Protein Synthesis)
140
Attachment of Preinitiation Complex
141
Scanning mRNA for AUG
142
rRNA and Proteins of Ribosomes

• Ribosomes are composed of both proteins and rRNA
• Confer some of the specificity of these complex
  interactions

143
Ribosomal Subunits
144
Protein Translation

• mRNA template
• Ribosomes peptidyl transferase
• tRNA adaptors

145
Protein Translation

• Amino-acyl tRNA synthetases specifically attach
  amino acids to tRNAs.
• amino acid ATP aminoacyl-AMP PPi
• aminoacyl-AMP tRNA aminoacyl-tRNA AMP

146
Protein Translation
147
Solving the Genetic Code

• Four nucleotides must code for 20 amino acids.
• 41 4, 42 16, 43 64, 44 256
• George Gamow

148
Solving the Genetic Code

• Synthetic RNAs
• UUUUUUUUU phe-phe-phe
• GGGGGGGGG gly-gly-gly
• CCCCCCCCC pro-pro-pro
• AAAAAAAAA lys-lys-lys
• Marshall Nirenberg and Johann Matthaei

149
Solving the Genetic Code

• Synthetic RNAs of defined sequence
• UCUCUC ser-leu-ser-leu
• Gobind Khorana
• Three nucleotides 1 codon 1 amino acid

150
The Genetic Code Redundancy And Wobble
151
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152
Structure of an Amino Acid
153
Amino Acids

• Nonpolar
• Alanine, Ala, A
• Isoleucine, Ile, I
• Leucine, Leu, L
• Methionine, Met, M
• Phenylalanine, Phe, F
• Tryptophan, Trp, W
• Valine, Val, V
• Negatively Charged (Acidic)
• Aspartic acid, Asp, D
• Glutamic acid, Glu, E

• Polar
• Asparagine, Asn, N
• Cysteine, Cys, C
• Glutamine, Gln, Q
• Glycine, Gly, G
• Proline, Pro, P
• Serine, Ser, S
• Threonine, Thr, T
• Tyrosine, Tyr, Y
• Positively Charged (Basic)
• Arginine, Arg, R
• Histidine, His, H
• Lysine, Lys, K

154
Amino Acid Structures
155
Isoelectric Point (pI)

• Amino acids are neutral at a pH, which is their
  isoelectric point (pI).

156
Peptide Bonds

• Amino acids are joined together by -C-C-N-
  linkages or peptide bonds to make proteins.

157
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158
INITIATION OF PROTEIN SYNTHESIS
159
Transfer Of Growing Chain
160
Transfer Of Growing Chain
161
Termination Of Chain
162
Protein TranslationTermination

• Termination of the amino acid chain is signaled
  by one of three nonsense, or termination codons,
  UAA, UAG, or UGA which are not charged with an
  amino acid.
• Termination or release factors trigger hydrolysis
  of the finished polypeptide from the final tRNA.

163
Location Of Translation Machinery
164
ENDOPLASMIC RETICULUM

• Microscopic series of tunnels
• Involved in transport and storage
• Two types of ER
• Rough ER (RER)
• Smooth ER (SER)

165
ENDOPLASMIC RETICULUM
166
Rough endoplasmic reticulum (RER)

• Originates from the outer membrane of the nuclear
  envelop
• Extends in a continuous network through cytoplasm
• Rough due to ribosomes
• Proteins are synthesized and shunted into the ER
  for packaging and transport
• First step in secretory pathway

167
Smooth Endoplasmic Reticulum (SER)

• Closed tubular network without ribosomes
• Functions in
• Nutrient processing
• Synthesis and storage of lipids, etc.

168
Rough Endoplasmic Reticulum (RER)
169
OVERVIEW OF SYNTHESIS
170
POLYRIBOSOMES
171
Protein Structure

• Primary amino acid sequence
• Secondary Intra-chain folding
• beta-pleated sheets
• alpha helices

172
Protein Structure

• Four levels of structure
• Primary
• Secondary
• Alpha helix, beta pleated sheet, random coil
• Tertiary
• Quaternary

173
Primary Structure Amino Acid Sequence
174
Secondary Structure Alpha Helix, Beta-pleated
Sheet, or Random Coil
175
Amino Acid Content Determines Protein Structure
and Function
176
Protein Structure

• Tertiary further folding, loss of which
  denatures protein
• Quaternary proteinprotein interaction for
  function.
• Monomers form multimers.
• Dimer
• Trimer
• Tetramer

177
Protein Function

• Enzymes
• Transport
• Storage
• Motility
• Structural
• Defense
• Regulatory

178
Conjugated Proteins

• Lipoproteinslipid
• Glycoproteinscarbohydrate
• Metalloproteinsmetal atoms
• Non-amino acid portionnonprotein prosthetic group

179
Modification Of Proteins
180
Modification Of Proteins
181
Post Translational Modification Of Proteins
182
Processing Of Insulin
183
Golgi Apparatus

• Consists of a stack of flattened sacs called
  cisternae
• Closely associated with ER
• Transitional vesicles from the ER containing
  proteins go to the Golgi apparatus for
  modification and maturation
• Condensing vesicles transport proteins to
  organelles or secretory proteins to the outside

184
Golgi Apparatus
185
Golgi Apparatus
186
Transport Process
187
Multiple Control Points
188
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189
Summary

• Proteins are made of combinations of 20 amino
  acids.
• Protein structure and function depends on the
  amino acid content and organization.
• A gene is defined, in part, by an open reading
  frame that contains the genetic code.
• In the genetic code, three nucleotides code for
  each amino acid.
• Proteins are translated from mRNA by peptidyl
  transferase activity in the ribosome, using tRNA
  as adaptors.