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Nucleic Acids Nucleic Acids Structures of Nucleic Acids DNA Replication RNA and Transcription 1 Nucleotides Nucleic acids consist of nucleotides that have a sugar, nitrogen base, and phosphate Base PO4 Sugar nucleoside 2 Nitrogen-Containing Bases O NH2 H N N N N NH2 N N H guanine (G) N H thymine (T) NH2 N N N O H adenine (A) O H CH3 CH3 N O N H cytosine (C) O H O CH3 N N H uracil (U)3 Sugars HOCH2 O OH ribose OH OH HOCH2 O OH OH (no O) deoxyribose 4 Nucleosides in DNA Base Adenine (A) Guanine (G) Cytosine (C) Thymine (T) Sugar Deoxyribose Deoxyribose Deoxyribose Deoxyribose Nucleoside Adenosine Guanosine Cytidine Thymidine 5 Nucleosides in RNA Base Adenine (A) Guanine (G) Cytosine (C) Uracil (U) Sugar ribose ribose ribose ribose Nucleoside Adenosine Guanosine Cytidine Uridine 6 Example of a Nucleoside NH2 N O O- O P O CH2 - N O O OH deoxyctyidine monophosphate (dCMP) 7 Nucleotides in DNA and RNA DNA dAMP dGMP dCMP dTMP Deoxyadenosine monophosphate Deoxyguanosine monophosphate Deoxycytidine monophosphate Deoxythymidine monophosphate RNA AMP GMP CMP UMP adenosine monophosphate guanosine monophosphate cytidine monophosphate uridine monophosphate 8 Structure of Nucleic Acids • • • • Polymers of four nucleotides Linked by alternating sugar-phosphate bonds RNA: ribose and A, G, C, U DNA: deoxyribose and A,G,C,T base P sugar nucleotide base P sugar base P sugar base P sugar nucleotide nucleotide nucleotide 9 Nucleic Acid Structure NH2 N CMP O O O- P O CH2 - O N O 3 NH2 3,5-phosphodiester bond OH O 5 O P O CH2 - N N N O N AMP O OH 10 Double Helix of DNA • DNA contains two strands of nucleotides • H bonds hold the two strands in a doublehelix structure • A helix structure is like a spiral stair case • Bases are always paired as A–T and G-C • Thus the bases along one strand complement the bases along the other 11 Complementary Base Pairs •Two H bonds for A-T •Three H bonds for G-C 12 Double Helix of DNA 13 Learning Check NA1 Write the complementary base sequence for the matching strand in the following DNA section: -A-G-T-C-C-A-A-T-G-C• • • • • • • • • • • • • • • • • • • • 14 Solution NA1 Write the complementary base sequence for the matching strand in the following DNA section: -A-G-T-C-C-A-A-T-G-C• • • • • • • • • • • • • • • • • • • • -T-C-A-G-G-T-T-A-C-G15 DNA Replication • DNA in the chromosomes replicates itself every cell division • Maintains correct genetic information • Two strands of DNA unwind • Each strand acts like a template • New bases pair with their complementary base • Two double helixes form that are copies of original DNA 16 DNA Unwinds G-C A-T C-G T-A GACT- -C -T -G -A 17 DNA Copied with Base Pairs Two copies of original DNA strand G-C A-T C-G T-A G-C A-T C-G G-A 18 Nucleic Acid Chemistry Where the info is…interpreting the blueprint Central Dogma Replicati on 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: GCAGUCAUGGGUAGGGAGGCAACCUGAACCGA C 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=u9dh O0iCLww 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