Nucleic Acid Chemistry

Where the info is…interpreting the blueprint

Central Dogma

DNA ---------------- RNA-------------- protein

Replication

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=u9dhO0iCLww

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 sequenceAUG 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 sequenceAUG 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 sequenceAUG GGU AGG GAG GCA ACC UGA ACC GACaa: G R E A T

New sequenceAUU GGU AGA GAG GCA ACC UGA ACC GACaa: 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 exampleOld sequence

AUG GGU AGG GAG GCA ACC UGA ACC GACaa: G R E A T

New sequenceAUG GGU AGG AGA GGC AAC CUG AAC CGA Caa: G R R G N L N R

Frame Shift Mutations

• Deletion example

• Old sequenceAUG 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 3’ GCCTAAGCTCA 5’

RNA 5’ CGGAUUCGAGU 3’

Back

Translation AnswerFind 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 codon…rest is garbage

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