nucleic acid chemistry

Nucleic Acid Chemistry

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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’


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

• In nucleus– nucleoli


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


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


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


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


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’


RNA Transcript

DNA 3’ GCCTAAGCTCA 5’

RNA 5’ CGGAUUCGAGU 3’


nucleic acid chemistry

Documents

dna strand

transcription dna template

dna codes

central dogma replication

replication leading

replica transcription

dna polymerase binds

replication helix