Download Nucleic Acid Chemistry

Nucleic Acid Chemistry
Where the info is…interpreting the
blueprint
Central Dogma
Replication
DNA ---------------- RNA-------------- protein
transcription
translation
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
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’
Replica
• Write the strand complementary to:
3’ ACTAGCCTAAGTCG 5’
Answer
Replication is Semiconservative
Replication
• Roles of enzymes
– Topoisomerases
– Helicase
– DNA polymerases
– ligase
• DNA binding proteins
– DNA synthesis
• Leading strand
• Lagging strand
Replication
Replication
• Helix opens
– Helicase
• Causes supercoiling upstream
– Topoisomerases (gyrase)
• DNA Binding Proteins
– Prevent reannealing
Replication
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
Replication
Replication
• Lagging strand
– 5’ end of template
• Can’t be made continuously as direction is wrong
– RNA primer
– New DNA made 5’  3’
• Opposite direction of replication
• Discontinuous
– Okazaki fragments
• Ligase closes gaps
Transcription
• DNA template made into RNA copy
– Uracil instead of Thymine
• One DNA strand is template
– Sense strand
• Other is just for replication
– Antisense (not to be confused with
nonsense!)
• In nucleus
– nucleoli
Transcription
• From following DNA strand, determine
RNA sequence
3’ GCCTAAGCTCA 5’
Answer
Transcription
Transcription
• DNA opens up
– Enzymes?
• RNA polymerase binds
– Which strand?
– Using DNA template, makes RNA
• 5’-3’
• Raw transcript called hnRNA
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
Introns and Exons
• Introns
– Intervening sequences
– Not all DNA codes for protein
– Regulatory info, “junk DNA”
• Exons
– Code for protein
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
Processing of hnRNA into mRNA
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
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
Genetic Code
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
Translation
• Steps:
– Find start codon (AUG)
– After start codon, read codons, in threes
– Use genetic code to translate
Translate the following:
GCAGUCAUGGGUAGGGAGGCAACCUGAACCGAC
Answer
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
Ribosome
Left is cartoon diagram
Right is actual picture
Transfer RNA
• Mostly double stranded
– Folds back on itself
• Several loops
– Anticodon loop
• Has complementary nucleotides to codons
• 3’ end where aa attach
Transfer RNA
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
Translation
Translation
• Happening of process (circa 1971)
• http://www.youtube.com/watch?v=u9dhO0
iCLww
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
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
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
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 codon…protein not made
Point Mutations
• Other possibilities,
– Stop codon inserted
• Truncated protein
– Stop codon changed
• Extra long protein
• Bottom line,
– Depends on what change is
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
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
Complementary DNA Strand
Template:
3’ ACTAGCCTAAGTCG 5’
5’ TGATCGGATTCAGC 3’
Back
RNA Transcript
DNA
RNA
Back
3’ GCCTAAGCTCA 5’
5’ CGGAUUCGAGU 3’
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
After stop codon…rest is garbage
Back
stop