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Nucleic Acids: Cell Overview and Core Topics. Outline Cellular Overview Anatomy of the Nucleic Acids Building blocks Structure (DNA, RNA ) Looking at the Central Dogma DNA Replication RNA Transcription Protein Synthesis. DNA and RNA in the Cell. Cellular Overview.

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Presentation Transcript
slide2

Outline

  • Cellular Overview
  • Anatomy of the Nucleic Acids
    • Building blocks
    • Structure (DNA, RNA)
  • Looking at the Central Dogma
    • DNA Replication
    • RNA Transcription
    • Protein Synthesis
slide4

Classes of Nucleic Acids: DNA

  • DNA is usually found in the nucleus
  • Small amounts are also found in:
    • mitochondria of eukaryotes
    • chloroplasts of plants
  • Packing of DNA:
    • 2-3 meters long
    • histones
  • genome = complete collection of hereditary information of an organism
slide5

Classes of Nucleic Acids: RNA

FOUR TYPES OF RNA

• mRNA - Messenger RNA• tRNA - Transfer RNA• rRNA - Ribosomal RNA• snRNA - Small nuclear RNA

slide7

Nucleic acids are linear polymers.

Each monomer consists of:

1. a sugar

2. a phosphate

3. a nitrogenous base

slide9

Nitrogenous Bases

DNA (deoxyribonucleic acid):

adenine (A) guanine (G)

cytosine (C) thymine (T)

Why ?

RNA (ribonucleic acid):

adenine (A) guanine (G)

cytosine (C) uracil (U)

slide10

Properties of purines and pyrimidines:

  • keto – enoltautomerism
  • strong UV absorbance
slide11

Pentoses of Nucleic Acids

This difference in structure affects secondary structure and stability.

Which is more stable?

slide12

Nucleosides

linkage of a base and a sugar.

slide13

Nucleotides

- nucleoside + phosphate

- monomers of nucleic acids

- NA are formed by 3’-to-5’ phosphodiester linkages

slide14

Shorthand notation:

  • sequence is read from 5’ to 3’
  • corresponds to the N to C terminal of proteins
slide16

Primary Structure

  • nucleotide sequences
slide17

Secondary Structure

DNA Double Helix

  • Maurice Wilkins and Rosalind Franklin
  • James Watson and Francis Crick
  • Features:
  • two helical polynucleotides coiled around an axis
  • chains run in opposite directions
  • sugar-phosphate backbone on the outside, bases on the inside
  • bases nearly perpendicular to the axis
  • repeats every 34 Å
  • 10 bases per turn of the helix
  • diameter of the helix is 20 Å
slide21

A and B forms are both right-handed double helix.

A-DNA has different characteristics from the more common B-DNA.

slide22

Z-DNA

  • left-handed
  • backbone phosphates zigzag
slide23

Comparison Between A, B, and Z DNA:

  • A-DNA: right-handed, short and broad, 11 bp per turn
  • B-DNA: right-handed, longer, thinner, 10 bp per turn
  • Z-DNA: left-handed, longest, thinnest, 12 bp per turn
slide25

Consequences of double helical structure:

  • 1. Facilitates accurate hereditary information transmission
  • Reversible melting
    • melting: dissociation of the double helix
    • melting temperature (Tm)
    • hypochromism
    • annealing
slide26

Tertiary Structure

Supercoiling

supercoiledDNA

relaxed DNA

slide32

Secondary Structure

transfer RNA (tRNA) : Brings amino acids to ribosomes during translation

slide33

Transfer RNA

  • Extensive H-bonding creates four double helical domains, three capped by loops, one by a stem
  • Only one tRNA structure (alone) is known
  • Many non-canonical base pairs found in tRNA
slide34

ribosomal RNA (rRNA) : Makes up the ribosomes, together with ribosomal proteins.

  • Ribosomes synthesize proteins
  • All ribosomes contain large and small subunits
  • rRNA molecules make up about 2/3 of ribosome
  • Secondary structure features seem to be conserved, whereas sequence is not
  • There must be common designs and functions that must be conserved
slide36

small nuclear RNA (snRNA) :With proteins, forms complexes that are used in RNA processing in eukaryotes. (Not found in prokaryotes.)

slide40

DNA Replication – process of producing identical copies of original DNA

    • strand separation followed by copying of each strand
    • fixed by base-pairing rules
slide45

DNA replication is bidirectional.

  • involves two replication forks that move in opposite direction
slide46

DNA Replication

  • Begins at specific start sites
    • in E. coli, origin of replication, oriC locus
    • binding site for dnaA, initiation protein
    • rich in A-T
slide63

Overall: each of the two DNA duplexes contain one “old” and one “new” DNA strand (semi-conservative) and half of the new strand was formed by leading strandand the other half by lagging strand.

slide64

DNA replication requires unwinding of the DNA helix.

    • expose single-stranded templates
    • DNA gyrase– acts to overcome torsional stress imposed upon unwinding
    • helicases– catalyze unwinding of double helix
      • disrupts H-bonding of the two strands
    • SSB (single-stranded DNA-binding proteins)– binds to the unwound strands, preventing re-annealing
slide65

Primer

RNA primes the synthesis of DNA.

Primase synthesizes short RNA.

slide66

DNA replication is semidiscontinuous

    • DNA polymerase synthesizes the new DNA strand only in a 5’3’ direction. Dilemma: how is 5’  3’ copied?
  • The leading strand copies continuously
  • The lagging strand copies in segments called Okazaki fragments (about 1000 nucleotides at a time) which will then be joined by DNA ligase
slide68

DNA Polymerase

= enzymes that replicate DNA

  • All DNA Polymerases share the following:
  • Incoming base selected in the active site (base-complementarity)
  • Chain growth 5’  3’ direction (antiparallel to template)
  • Cannot initiate DNA synthesis de novo (requires primer)

First DNA Polymerase discovered – E.coli DNA Polymerase I (by Arthur Kornberg and colleagues)

Arthur Kornberg

1959 Nobel Prize in Physiology and Medicine

Roger D. Kornberg

2006 Nobel Prize in Chemistry

http://www.nobelprize.org

slide69

DNA Polymerase

  • specificity dictated by H-bonding and shape complementarity between bases
    • binding of correct base is favorable (more stable)
    • interaction of residues in the enzyme to the minor groove of DNA
    • close down around the incoming NTP
slide72

3’  5’ exonuclease activity

- removes incorrect nucleotides from the 3’-end of the growing chain (proofreader and editor)

- polymerase cannot elongate an improperly base-paired terminus

  • proofreading mechanisms
    • Klenow fragment – removes mismatched nucleotides from the 3’’ end of DNA (exonuclease activity)
    • detection of incorrect base
      • incorrect pairing with the template (weak H-bonding)
      • unable to interact with the minor groove (enzyme stalls)
slide73

Exonuclease activity

5’  3’ exonuclease activity

  • remove distorted segments lying in the path of the advancing polymerase
slide74

DNA Ligase

= seals the nicks between Okazaki fragments

  • DNA ligase seals breaks in the double stranded DNA
  • DNA ligases use an energy source (ATP in eukaryotes and archaea, NAD+ in bacteria) to form a phosphodiester bond between the 3’ hydroxyl group at the end of one DNA chain and 5’-phosphate group at the end of the other.
slide76

DNA replication terminates at the Ter region.

    • the oppositely moving replication forks meet here and replication is terminated
    • contain core elements 5’-GTGTGTTGT
    • binds termination protein (Tus protein)
slide77

Eukaryotic DNA Replication

  • Like E. coli, but more complex
  • Human cell: 6 billion base pairs of DNA to copy
  • Multiple origins of replication: 1 per 3000-30000 base pairs
  • E.coli 1 chromosome
  • Human 23
  • E.coli circular chromosome;
  • Human linear
slide78

Telomeres

The Ends of Linear DNA Possess Telomeres

  • Present because DNA is shortened after each round of replication
  • Contains hundreds of tandem repeats of a hexanucleotide sequence (AGGGTT in humans)
  • Telomeres at the 3’ end is G rich and is slightly longer
  • May form large loops to protect chromosome ends
slide80

DNA Recombination =

natural process of genetic rearrangement

  • recombinases
  • Holliday junction – crosslike structure
slide81

Mutations

  • Substitution of base pair
    • transition
    • transversion
  • Deletion of base pair/s
  • Insertion/Addition of base pair/s

Macrolesions: Mutations involving changes in large portions of the genome

DNA replication error rate: 3 bp during copying of 6 billion bp

slide82

Agents of Mutations

  • Physical Agents
    • UV Light
    • Ionizing Radiation
  • Chemical Agents
    • Some chemical agents can be classified further into
    • Alkylating
    • Intercalating
    • Deaminating
  • Viral
slide83

UV Light Causes Pyrimidine Dimerization

  • Replication and gene expression are blocked
slide85

Chemical mutagens

    • 5-bromouracil and 2-aminopurine can be incorporated into DNA
slide86

Deaminating agents

    • Ex: Nitrous acid (HNO2)
    • Converts adenine to hypoxanthine, cytosine to uracil, and guanine to xanthine
    • Causes A-T to G-C transitions
slide90

Acridines

    • Intercalate in DNA, leading to insertion or deletion
    • The reading frame during translation is changed
slide91

DNA Repair

  • Direct repair
    • Photolyase cleave pyrimidine dimers
  • Base excision repair
    • E. coli enzyme AlkA removes modified bases such as 3-methyladenine (glycosylase activity is present)
  • Nucleotide excision repair
    • Excision of pyrimidine dimers (need different enzymes for detection, excision, and repair synthesis)
slide93

QUIZ

  • Draw the structure of any nitrogenous base of your picking. (1 pt)
  • What is the difference between the glycosidic bond and the phosphodiester bond? (2 pts)
  • Give the reason why DNA utilizes the deoxyribose while RNA uses the ribose. (2 pts)
  • Enumerate all the enzymes and proteins involved in DNA replication and briefly state their importance/function. A short concise answer will suffice. (4 pts)
  • Give the partner strand of this piece of DNA:
    • 5-ACTCATGATTAGCAG-3  (1 pt)
slide95

Process of Transcription has four stages:

  • Binding of RNA polymerase at promoter sites
  • Initiation of polymerization
  • Chain elongation
  • Chain termination
slide96

Transcription (RNA Synthesis)

  • RNA Polymerases
    • Template (DNA)
    • Activated precursors (NTP)
    • Divalent metal ion (Mg2+ or Mn2+)
  • Mechanism is similar to DNA Synthesis
slide97

Reece R. Analysis of Genes and Genomes.2004. p47.

  • Limitations of RNAP II:
  • It can’t recognize its target promoter and gene. (BLIND)
  • It is unable to regulate mRNA production in response to developmental and environmental signals. (INSENSITIVE)
slide98

Start of Transcription

  • Promoter Sites
    • Where RNA Polymerase can indirectly bind
slide99

Preinitiation Complex (PIC)

TATA box – a DNA sequence (5’—TATAA—3’) found in the promoter region of most eukaryotic genes.

Abeles F, et al. Biochemistry. 1992. p391.

Transcription Factors (TF):

Hampsey M. Molecular Genetics of RNAP. Microbiology and Molecular Biology Reviews. 1998. p7.

slide106

Termination of Transcription

1. Intrinsic termination = termination sites

  • Terminator Sequence
    • Encodes the termination signal
    • In E. coli – base paired hair pin (rich in GC) followed by UUU…

causes the RNAP to pause

causes the RNA strand to detach from the DNA template

slide107

Termination of Transcription

2. Rho termination = Rho protein, ρ

slide108

prokaryotes: transcription and translation happen in cytoplasm

eukaryotes: transcription (nucleus); translation (ribosome in cytoplasm)

slide109

In eukaryotes, mRNA is modified after transcription

    • Capping, methylation
    • Poly-(A) tail
    • splicing

capping: guanylyl residue

capping and methylation ensure stability of the mRNA template; resistance to exonuclease activity

slide111

Introns & Exons

  • Introns
    • Intervening sequences
  • Exons
    • Expressed sequences
slide112

Splicing

Spliceosome: multicomponent complex of small nuclear ribonucleoproteins (snRNPs)

splicing occurs in the spliceosome!

slide113

Reverse Transcription

  • RNA-Directed DNA Polymerase
  • 1964: Howard Temin notices that DNA synthesis inhibitors prevent infection of cells in culture by RNA tumor viruses. Temin predicts that DNA is an intermediate in RNA tumor virus replication
  • 1970: Temin and David Baltimore (separately) discover the RNA-directed DNA polymerase - aka "reverse transcriptase"
slide114

Reverse Transcriptase

  • Primer required, but a strange one - a tRNA molecule that the virus captures from the host
  • RT transcribes the RNA template into a complementary DNA (cDNA) to form a DNA:RNA hybrid
  • All RNA tumor viruses contain a reverse transcriptase
slide115

RT II

  • Three enzyme activities
    • RNA-directed DNA polymerase
    • RNase H activity - degrades RNA in the DNA:RNA hybrids
    • DNA-directed DNA polymerase - which makes a DNA duplex after RNase H activity destroys the viral genome
  • HIV RT: very error-prone (1 bp /2000 to 4000 bp)
  • HIV therapy: AZT (or 3\'-azido-2\',3\'- dideoxythymidine) specifically inhibits RT
slide117

Translation

Starring three types of RNA

  • mRNA
  • tRNA
  • rRNA
slide118

Properties of mRNA

  • In translation, mRNA is read in groups of bases called “codons”
  • One codon is made up of 3 nucleotides from 5’ to 3’ of mRNA
  • There are 64 possible codons
  • Each codon stands for a specific amino acid, corresponding to the genetic code
  • However, one amino acid has many possible codons. This property is termed degeneracy
  • 3 of the 64 codons are terminator codons, which signal the end of translation
slide119

Genetic Code

  • 3 nucleotides (codon) encode an amino acid
  • The code is nonoverlapping
  • The code has no punctuation
slide121

Synonyms

  • Different codons, same amino acid
  • Most differ by the last base
    • XYC & XYU
    • XYG & XYA
  • Minimizes the deleterious effect of mutation
slide122

Practice

  • Encoded sequences.
  • (a) Write the sequence of the mRNA molecule synthesized from a DNA template strand having the sequence
  • (b) What amino acid sequence is encoded by the following base sequence of an mRNA molecule? Assume that the reading frame starts at the 5 end.
slide123

Answers

  • (a) 5’ -UAACGGUACGAU-3’ .
  • (b) Leu-Pro-Ser-Asp-Trp-Met.
slide124

tRNA as Adaptor Molecules

  • Amino acid attachment site
  • Template recognition site
    • Anticodon
      • Recognizes codon in mRNA
slide132

Mechanics of Protein Synthesis

  • All protein synthesis involves three phases: initiation, elongation, termination
  • Initiation involves binding of mRNA and initiator aminoacyl-tRNA to small subunit(30S), followed by binding of large subunit (50S) of the ribosome
  • Elongation: synthesis of all peptide bonds - with tRNAs bound to acceptor (A) and peptidyl (P) sites.
  • Terminationoccurs when "stop codon" reached
slide133

Translation

  • Occurs in the ribosome
  • Prokaryote START
    • fMet (formylmethionine) bound to
    • initiator tRNA
    • Recognizes AUG and sometimes
    • GUG (but they also code for Met
    • and Val respectively)
    • AUG (or GUG) only part of the initiation signal; preceded by a purine-rich sequence
slide134

Translation

  • Eukaryote START
    • AUG nearest the 5’ end is usually the start signal
slide138

Termination

  • Stop signals (UAA, UGA, UAG):
    • recognized by release factors (RFs)
    • hydrolysis of ester bond between polypeptide and tRNA
slide139

Reference:

Garrett, R. and C. Grisham. Biochemistry. 3rd edition. 2005.

Berg, JM, Tymoczko, JL and L. Stryer. Biochemistry. 5th edition. 2002.

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