Nucleic Acids: Cell Overview and Core Topics
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Transcript of Nucleic Acids: Cell Overview and Core Topics
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Nucleic Acids: Cell Overview and Core Topics
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Cellular OverviewDNA and RNA in the Cell
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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
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Classes of Nucleic Acids: RNA
FOUR TYPES OF RNA
• mRNA - Messenger RNA
• tRNA - Transfer RNA
• rRNA - Ribosomal RNA
• snRNA - Small nuclear RNA
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Anatomy of Nucleic Acids
THE BUILDING BLOCKS
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Nucleic acids are linear polymers.
Each monomer consists of:1. a sugar2. a phosphate3. a nitrogenous base
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Nitrogenous Bases
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Nitrogenous Bases
DNA (deoxyribonucleic acid):adenine (A) guanine (G)cytosine (C) thymine (T)
RNA (ribonucleic acid):adenine (A) guanine (G)cytosine (C) uracil (U)
Why ?
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Pentoses of Nucleic Acids
This difference in structure affects secondary structure and stability.
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Nucleosideslinkage of a base and a sugar.
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Nucleotides- nucleoside + phosphate
- monomers of nucleic acids - NA are formed by 3’-to-5’ phosphodiester linkages
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Shorthand notation:
- sequence is read from 5’ to 3’- corresponds to the N to C terminal of
proteins
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Nucleic Acids: Structure
DNA
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Primary Structure• nucleotide sequences
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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
Secondary Structure
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Double helix stabilized by hydrogen bonds.
Which is more stable?
ATCTGGCATTAGACCGTA
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Axial view of DNA
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A and B forms are both right-handed double helix.
A-DNA has different characteristics from the more common B-DNA.
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• left-handed• backbone phosphates zigzag
Z-DNA
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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
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Supercoiling
relaxed DNA
supercoiled DNA
Tertiary Structure
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Consequences of double helical structure:
1. Facilitates accurate hereditary information transmission
2.Reversible melting• melting: dissociation of the double helix• melting temperature (Tm)• hypochromism• annealing
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Structure of Single-stranded DNA
Stem Loop
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Nucleic Acids: Structure
RNA
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Secondary Structure
transfer RNA (tRNA) : Brings amino acids to
ribosomes during translation
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ribosomal RNA (rRNA) : Makes up the ribosomes, together with ribosomal proteins.
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messenger RNA (mRNA) : Encodes amino acid sequence of a polypeptide
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small nuclear RNA (snRNA) :With proteins, forms complexes that are used in RNA processing in eukaryotes. (Not found in prokaryotes.)
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Central Dogma
DNA Replication, Recombination, and Repair
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Central Dogma
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DNA Replication – process of producing identical copies of original DNA
• strand separation followed by copying of each strand
• fixed by base-pairing rules
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DNA replication is bidirectional. involves two replication forks that move in opposite
direction
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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
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Primer
RNA primes the synthesis of DNA.
Primase synthesizes short RNA.
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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
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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.
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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)
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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 chromosomeHuman 23E.coli circular chromosome; Human linear
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Mutations1. Substitution of base
paira. transitionb. transversion
2. Deletion of base pair/s
3. Insertion/Addition of base pair/s
DNA replication error rate: 3 bp during copying of 6 billion bp
Macrolesions: Mutations involving changes in large portions of the genome
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Agents of Mutations1. Physical Agents
a) UV Lightb) Ionizing Radiation
2. Chemical AgentsSome chemical agents can be
classified further intoa) Alkylatingb) Intercalatingc) Deaminating
3. Viral
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DNA Repair
Direct repairPhotolyase cleave pyrimidine dimers
Base excision repairE. coli enzyme AlkA removes modified bases
such as 3-methyladenine (glycosylase activity is present)
Nucleotide excision repairExcision of pyrimidine dimers (need different
enzymes for detection, excision, and repair synthesis)
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Central DogmaRNA Transcription
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Process of Transcription has four stages:
1. Binding of RNA polymerase at promoter sites2. Initiation of RNA polymerization3. Chain elongation4. Chain termination
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Transcription (RNA Synthesis)
RNA PolymerasesTemplate (DNA)Activated precursors (NTP)Divalent metal ion (Mg2+ or Mn2+)
Mechanism is similar to DNA Synthesis
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Start of TranscriptionPromoter Sites
Where RNA Polymerase can indirectly bind
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Termination of Transcription
Terminator SequenceEncodes the
termination signalIn E. coli – base
paired hair pin (rich in GC) followed by UUU…
1. Intrinsic termination = termination sites
causes the RNAP to pause
causes the RNA strand to detach from the DNA template
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Termination of Transcription
2. Rho termination = Rho protein, ρ
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prokaryotes: transcription and translation happen in cytoplasm
eukaryotes: transcription (nucleus); translation (ribosome in cytoplasm)
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In eukaryotes, mRNA is modified after transcriptionCapping, methylationPoly-(A) tail, splicing
capping: guanylyl residue
capping and methylation ensure stability of the mRNA template; resistance to exonuclease activity
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Eukaryotic genes are split genes: coding regions (exons) and noncoding regions (introns)
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Introns & Exons
IntronsIntervening
sequencesExons
Expressed sequences
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Splicing
Spliceosome: multicomponent complex of small nuclear ribonucleoproteins (snRNPs)
splicing occurs in the spliceosome!
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Central DogmaTranslation: Protein Synthesis
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TranslationStarring three types of RNA
1.mRNA
2.tRNA
3.rRNA
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Properties of mRNA1. In translation, mRNA is read in groups of bases called “codons”
2. One codon is made up of 3 nucleotides from 5’ to 3’ of mRNA
3. There are 64 possible codons
4. Each codon stands for a specific amino acid, corresponding to the genetic code
5. However, one amino acid has many possible codons. This property is termed degeneracy
6. 3 of the 64 codons are terminator codons, which signal the end of translation
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Genetic Code
3 nucleotides (codon) encode an amino acid
The code is nonoverlappingThe code has no punctuation
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Synonyms
Different codons, same amino acidMost differ by the last base
XYC & XYU XYG & XYA
Minimizes the deleterious effect of mutation
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tRNA as Adaptor Molecules
Amino acid attachment site
Template recognition siteAnticodon
Recognizes codon in mRNA
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tRNA as Adaptor Molecules
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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.
Termination occurs when "stop codon" reached
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TranslationOccurs in the ribosomeProkaryote 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
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Translation
Eukaryote START
AUG nearest the 5’ end is usually the start signal
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Termination
Stop signals (UAA, UGA, UAG):• recognized by release factors (RFs)• hydrolysis of ester bond between polypeptide and
tRNA
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Reference:
Garrett, R. and C. Grisham. Biochemistry. 3rd edition. 2005.
Berg, JM, Tymoczko, JL and L. Stryer. Biochemistry. 5th edition. 2002.