Nucleic Acid Chemistry

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Nucleic Acid Chemistry

• Where the info is

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Central Dogma

• DNA ----------------? RNA--------------?
  protein

Replication
transcription
translation
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Central Dogma

• Replication
• DNA making a copy of itself
• Making a replica
• Transcription
• DNA being made into RNA
• Still in nucleotide language
• Translation
• RNA being made into protein
• Change to amino acid language

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Replication

• Remember that DNA is self complementary
• Replication is semiconservative
• One strand goes to next generation
• Other is new
• Each strand is a template for the other
• If one strand is 5 AGCT 3
• Other is 3 TCGA 5

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Replica

• Write the strand complementary to
• 3 ACTAGCCTAAGTCG 5
• Answer

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Replication is Semiconservative
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Replication

• Roles of enzymes
• Topoisomerases
• Helicase
• DNA polymerases
• ligase
• DNA binding proteins
• DNA synthesis
• Leading strand
• Lagging strand

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Replication
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Replication

• Helix opens
• Helicase
• Causes supercoiling upstream
• Topoisomerases (gyrase)
• DNA Binding Proteins
• Prevent reannealing

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Replication
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Replication

• Leading strand
• 3 end of template
• As opens up, DNA polymerase binds
• Makes new DNA 5 -? 3
• Same direction as opening of helix
• Made continuously

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Replication
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Replication

• Lagging strand
• 5 end of template
• Cant be made continuously as direction is wrong
• RNA primer
• New DNA made 5 ? 3
• Opposite direction of replication
• Discontinuous
• Okazaki fragments
• Ligase closes gaps

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Transcription

• DNA template made into RNA copy
• Uracil instead of Thymine
• One DNA strand is template
• Sense strand
• Other is just for replication
• Antisense
• In nucleus
• nucleoli

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Transcription

• From following DNA strand, determine RNA sequence
• 3 GCCTAAGCTCA 5
• Answer

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Transcription
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Transcription

• DNA opens up
• Enzymes?
• RNA polymerase binds
• Which strand?
• Using DNA template, makes RNA
• 5-?3
• Raw transcript called hnRNA

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Transcription

• How does RNA polymerase know where to start?
• upstream promotor sequences
• Pribnow Box
• TATA box
• RNA polymerase starts transcription X
  nucleotides downstream of TATA box

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Introns and Exons

• Introns
• Intervening sequences
• Not all DNA codes for protein
• Regulatory info, junk DNA
• Exons
• Code for protein

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Processing of hnRNA into mRNA

• 3 steps
• Introns removed
• Self splicing
• 5 methyl guanosine cap added
• Poly A tail added
• Moved to cytosol for translation

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Processing of hnRNA into mRNA
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Translation

• RNA --? Protein
• Change from nucleotide language to amino acid
  language
• On ribosomes
• Vectorial nature preserved
• 5 end of mRNA becomes amino terminus of protein
• Translation depends on genetic code

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Genetic Code

• Nucleotides read in triplet codons
• 5 -? 3
• Each codon translates to an amino acid
• 64 possible codons
• 3 positions and 4 possiblities (AGCU) makes 43 or
  64 possibilities
• Degeneracy or redundancy of code
• Only 20 amino acids
• Implications for mutations

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Genetic Code
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Genetic Code

• Not everything translated
• AUG is start codon
• Find the start codon
• Also are stop codons
• To determine aa sequence
• Find start codon
• Read in threes
• Continue to stop codon

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Translation

• Steps
• Find start codon (AUG)
• After start codon, read codons, in threes
• Use genetic code to translate
• Translate the following
• GCAGUCAUGGGUAGGGAGGCAACCUGAACCGAC
• Answer

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Translation Process

• Requires Ribosomes, rRNA, tRNA and, of course,
  mRNA
• Ribosome
• Made of protein and rRNA
• 2 subunits
• Has internal sites for 2 transfer RNA molecules

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Ribosome

• Left is cartoon diagram Right is actual
  picture

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Transfer RNA

• Mostly double stranded
• Folds back on itself
• Several loops
• Anticodon loop
• Has complementary nucleotides to codons
• 3 end where aa attach

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Transfer RNA
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Translation

• Initiation
• Ribosomal subunits assemble on mRNA
• rRNA aids in binding of mRNA
• Elongation
• tRNAs with appropriate anticodon loops bind to
  complex
• have aa attached (done by other enzymes)
• Amino acids transfer form tRNA 2 to tRNA 1
• Process repeats
• Termination
• tRNA with stop codon binds into ribosome
• No aa attached to tRNA
• Complex falls apart

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Translation
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Translation

• Happening of process (circa 1971)
• http//www.youtube.com/watch?vu9dhO0iCLww

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Mutations

• Changes in nucleotide sequence
• Can cause changes in aa sequence
• Degeneracy in genetic code can prevent
• Two types
• Point mutations
• Single nucleotide changes
• Frame shift
• Insertions or deletions

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Point Mutations

• Single nucleotide changes
• Old sequence
• AUG GGU AGG GAG GCA ACC UGA ACC GAC
• aa G R E A T
• New sequence
• AUG GGU AGU GAG GCA ACC UGA ACC GAC
• aa G S E A T

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Point mutations

• Depending on change, may not change aa sequence
• Old sequence
• AUG GGU AGG GAG GCA ACC UGA ACC GAC
• aa G R E A T
• New sequence
• AUG GGU AGA GAG GCA ACC UGA ACC GAC
• aa G R E A T

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Point Mutations

• Change could make little difference
• If valine changed to leucine, both nonpolar
• Change could be huge,
• Could erase start codon
• Old sequence
• AUG GGU AGG GAG GCA ACC UGA ACC GAC
• aa G R E A T
• New sequence
• AUU GGU AGA GAG GCA ACC UGA ACC GAC
• aa no start codonprotein not made

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Point Mutations

• Other possibilities,
• Stop codon inserted
• Truncated protein
• Stop codon changed
• Extra long protein
• Bottom line,
• Depends on what change is

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Frame Shift mutations

• Insertions or deletions
• Change the reading frame
• Insertion example
• Old sequence
• AUG GGU AGG GAG GCA ACC UGA ACC GAC
• aa G R E A T
• New sequence
• AUG GGU AGG AGA GGC AAC CUG AAC CGA C
• aa G R R G N L N R

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Frame Shift Mutations

• Deletion example
• Old sequence
• AUG GGU AGG GAG GCA ACC UGA ACC GAC
• aa G R E A T
• New sequence Delete second A (Underlined above)
• AUG GGU GGG AGG CAA CCU GAA CCG AC
• aa G G R Q P G P

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Complementary DNA Strand

• Template
• 3 ACTAGCCTAAGTCG 5
• 5 TGATCGGATTCAGC 3
• Back

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RNA Transcript

• DNA 3 GCCTAAGCTCA 5
• RNA 5 CGGAUUCGAGU 3
• Back

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Translation Answer

• Find start codon
• GCAGUCAUGGGUAGGGAGGCAACCUGAACCGAC
• Read in threes after that
• AUG GGU AGG GAG GCA ACC UGA ACC GAC
• Using Genetic code
• AUG GGU AGG GAG GCA ACC UGA ACC GAC
• G R E A T stop
• After stop codonrest is garbage
• Back