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Nucleic Acids, DNA, RNA and Protein Synthesis CHM 341 Suroviec Fall 2016 I. Nucleotides, Nucleic Acids and Bases A. Bases • Planar, aromatic, heterocyclic • Purine (2 rings) • Pyrimidine (1 ring) Adenine (A) Guanine (G) Thyamine (T) Cytosine ( C) Uracil (U) B. Nucleosides • Ribonucleotides – sugar = ribose • Deoxyriboneculeotide – Sugar = 2´-deoxyribose C. Nucleotides (total molecule) • Have a phosphate on carbon #5 • Can have up to 3 phosphates • Monophosphate (NMP) • Diphosphate (NDP) • Triphosphate (NTP) – Where N is any one of the nucleic acids II. Nucleic Acid Structure • Can be found singly • Most often found in a polymer • DNA (or RNA) polymerizes 5´ phosphate to 3´ OH – Makes phosphodiester bond • Polymer of non-identical residues has a property that individual monomers do not. A. Base composition of DNA • 1940’s Erwin Chargaff discovered that when measuring the amount of each base A = T and G = C. Lead to Chargaff’s rules. • Watson and Crick took this data and other material that hinted that DNA stacked to propose that DNA was double stranded. • Maurie Wilkins and Rosalind Franklin made and X-ray that indicated that DNA was helical in nature • Put the bases together in such a way so that the complimentary Hbonds were formed and the width of the base pairs would be similar. Characteristics of DNA model 1. 2. 3. 4. 5. 6. DNA strands run in opposite directions (antiparallel) Sugar phosphate backbone is found on outside, bases inside and pair up Each base is H-bonded with a base on the opposite strand with the same number of H-bonds A complete turn takes 34 Å and has 10 bases per turn 2 helical polynucleotide chains coiled around a central axis (diameter = 20 Å) DNA strand is quite stiff and will not bend much around the axis - DNA Helix is right handed III. Overview of Nucleic Acid Function • Carries genetic info • Directs protein synthesis • Double stranded nature allows for easy replication A. • DNA replication • W-C model allows each DNA strand to act as template for replication 2 hypothesis for replication came forth: • Conservative: where the parental DNA strand retains both old stands and creates new ds DNA • Semi conservative: where the created DNA has one old strand and one new strand. Shown to be how DNA replicated DNA, RNA & Protein Synthesis • DNA directs its own replication and is also transcribed into RNA. RNA then translates into proteins. – CENTRAL DOGMA of MOLECULAR BIOLOGY • Transcription: transferring into from DNA --> RNA • Translation: transferring info from RNA --> proteins IV. Replication • Involves 20+ proteins – Helicases: opens the double strand, splits the strands apart starting at replication fork rich in A-T – SSB: bind to the single strand DNA stablizing it – Primase: adds short stretches of RNA and allows the the DNA polymerase to start. – DNA polymerase I: catalyzes the addition of deoxynucleotides to the chain – DNA Polymerase I & III: add DNA with high fidelity to the newly growing DNA strand. – Ligase: closes up gaps in the DNA DNA polymerase I • DNA polymerase I catalyzes addition of a addition of dNTP to chain • Requires dATP, dGTP, TTp, dCTP and Mg2+ • Elongation occurs 5´ to 3´ where 3´ hydroxyl bind to the new deoxyribonucleotide • DNA polymerase is a “template directed enzyme” DNA polymerase III • Adds nucleotides to the 3´ end of the chain • New strand reads 5´ to 3´ • Needs a primer with free 3´ hydroxyl group to start addition of new DNA – The strand is going to be started with a RNA primer that is later removed and replaced • The incoming dNTP first forms an appropriate base pair and then the DNA polymerase III links the incoming bases together • Binds complementary DNA nucleotides starting at the 3´ end of the RNA primer at a rate of 1000/second • Makes a mistake 1/108 IV. Replication • Opening of the DNA – Double stranded DNA is opened by helicase – Kept open by SSB – Exposed DNA bind DNA polymerase III and RNA synthesizing protein primase • This makes the replication fork IV. Replication • Leading strand synthesis begins with synthesis of primase of short RNA primer • dNTPs are added by DNA polymerase III • Continously added to this strand toward the fork Replication • Lagging strand synthesis is done in short bursts • Needs multiple RNA primers • Synthesized in opposite direction of the fork • DNA primase moves 5’ to 3’ and makes RNA primer to which DNA is then added by DNA polymerase III Replication • Keeping the DNA sequence correct is important: 1 mispair per 109 base pairs • Polymerase reaction occurs in 2 stages – Incoming dNTP base pairs with the template while enzyme is open catalytically inactive – Polymerization only occurs after polymerase has closed around base pair which positions residues Transcription • DNA is in the nucleus • Protein synthesis takes place in the ribosome • RNA is the intermediate • Cells contain 3 types of RNA – Ribosomal RNA (rRNA) – Transfer RNA (tRNA) – Messenger RNA (mRNA) RNA polymerase • RNAP couples together the ribonucleotide triphosphates on DNA templates • Builds RNA in the 5’ 3’ direction (reads the DNA in the 3’ --> 5’ direction) RNA polymerase • 3’ hydroxyl group attacks the triphosphate • Creates phosphodiester bond • Releases PPi • Does not need a primer RNA polymerase • Initiation of RNA synthesis occurs only at promoters – Usually starts at GTP or ATP – New RNA strand base pairs temporarily with DNA template to form DNA/RNA template – DNA must unwind then rewind – Template strand – Nontemplate strand or coding strand RNA polymerase • RNA polymerase lacks ability to proof read • No 3’--> 5’ exonuclease activity • One error in 104 ribonucleotides added Post transcription of RNA • In Eukaryotes RNA is further modified • mRNA undergoes gene splicing where introns are removed and exons are rejoined • 5’ obtains a cap • 3’ gets polyA tail Characteristics of RNA • Contains AUGC • Uracil is less “energy expensive” • Normally single stranded • Has –OH on 2’ carbon of ribose • 1. 2. 3. 4. 5. 6. 7. Seven roles of RNA mRNA – carries DNA code to make proteins rRNA – forms complex of 2/3 RNA, 1/3 protein to form protein in ribosome tRNA – carries the amino acids to the mRNA snRNA – helps splice exons Ribozymes – RNA capable of catalytic activity Antisense RNA – act to bind RNA to stop translation Viral RNA – carry hereditary information Translation • mRNA to proteins – Need mRNA, ribosome and tRNA • mRNA is produced from DNA • mRNA read from ribosomes and tRNA Ribosome • Large protein/RNA complex • 2 units (large/small) • Synthesis begins at start codon near 5’ end – Smaller unit (usually has tRNA bound) binds to AUG codon on mRNA – binds to large subunit – Large unit then binds – Large unit has 3 tRNA binding sites (APE) – A: aminoacyl-tRNA – P:peptidyl-tRNA – E: free-tRNA Initiation • AUG signals the beginning of polypeptide chains • Read the code off of the mRNA and translate into amino acids • One start codon • 3 stop codon tRNA • Read the code on the mRNA and translate into the correct amino acid • Acceptor stem – 5’ terminal nucleotide and 3’ terminal nucleotide (-OH group where amino acid binds) – 3’end always has CCA sequence • Specific linkage is catalyzed by amino acyl-tRNA synthetase (tranferase). • Anticodon recognizes the complementary codon on the mRNA Aminoacylation • Process of adding an aminoacyl group to a compound – Produces tRNA molecules with their CCA 3’ ends covalently linked to an amino acid • Aminoacyl tRNA synthetase (one specific for each amino acid) • Needs ATP to drive the reaction Initiation and Elongation • mRNA bearing the code for the polypeptide binds to the small ribosome unit • Aminoacyl-tRNA then binds followed by larger ribosomal unit • Aminoacyl-tRNA base-pairs with mRNA codon AUG to start the polypeptide • Chain is elongated by addition of amino acids • Added by individual tRNA • Polypeptides are grown from amino-terminal end to carboxyl-end Elongation • • • • • mRNA passes through ribosome AUG is held in P site 2nd amino acid binds in the A site Make peptide bond Ribosome then moves toward 3’ end using GTP and leaving A site open