nucleic acid chemistry where the info is…interpreting the blueprint
TRANSCRIPT
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
Back