BCH 380 1st edition Final Exam Study Guide Lecture 33 April 20 Nucleic Acids Know nomenclature for base nucleoside nucleotide oligonucleotide Nucleosides o o o o Nucleic acid base plus sugar sugar is pentose Ribose or deoxyribose Ribonucleoside or deoxyribonucleoside Base is linked to sugar by N glycosidic bond versus O glycosidic bond in carbohydratesGlycosidic bond is always beta Nucleosides are named by adding idine to pyrimidine names and osine to purine names Sugar numbering uses the to distinguish from base numbering Note beta conformation about anomeric carbon Ribonucleosides Purines Guanosine Adenosine Pyrimidines Ribothymidine Cytidine Uridine Deoxyribonucleosides Purines 2 deoxguanosine 2 deoxyadenosine Pyrimidines Thymidine 2 deoxycytidine 2 deoxyuridine Nucleotides Nucleotide is Sugar Base Phosphate Naming can be written out or annotated 5 triphosphate 2 deoxyguanosine 5 dGTP Uridine 3 monophosphate 3 rUMP 3 5 cyclic Adenosine Monophosphate 3 5 cAMP NTP s where N is any or unknown base are carriers of energy through phosphate or pyrophosphate transfer ATP general energy carrier GTP energy for protein synthesis CTP energy for phospholipid synthesis UTP energy for carbohydrate synthesis Almost always bound to Mg O2 of pyrimidines over plane of sugar in synH 8 of purine over plane of sugar in anti Basic structural properties of B form DNA duplex versus A or Z form H bonding patterns between bases in duplex DNA The all sp2 hybridization of the bases make them planar Important for stacking in DNA Substituents hanging off the rings is what makes them base pair differently Keto form predominates at physiological pH Free Rotation in nucleosides and nucleotides somewhat Needs to be anti conformation for base pairing in duplex B form DNA The linkage is made from the 3 alcohol of the sugar ribose or deoxyribose of one nucleotide to the 5 phosphate of another sugar o The directionality of the nucleic acid polymer is thus 5 3 o Defines the reading frame much like N terminus to C terminus in proteins o Naming is similar to others dinucleotide trinucleotide etc o Oligonucleotide 10 nt to polynucleotide The polymer forms 3 5 with release of pyrophosphate from incoming NTP and phosphodiester bond formation This would be 5 d TpApCpG 3 or 5 TACG 3 Double stranded DNA is connected via base pairing with hydrogen bonds Nucleosomes form beads on a string Histones are highly positively charged o Lots of R and K o They form an octamer core For DNA to wrap around 3 Types of RNA o mRNA messenger RNA o tRNA transfer amino acids o rRNA ribosomal RNA 3 end amino acid is attached as an acyl ester Three nucleotides at the bottom match up to each codon codon anticodon pairing Lecture 34 April 22 DNA Replication What is replication and what does it produce Replication of DNA produces two identical copies of the original DNA which means that high fidelity is necessary for accurate transmission of genetic information The basics of DNA replication involves strand separation unwinding with each strand serving as a template and with base pairing dictating the accurate replication Know the enzymes and their roles in replication 1 DNA Gyrase a Type II topoisomerase that introduces negative supercoils to overcome the torsional stress of unwinding Requires ATP hydrolysis for energy 2 Helicase ATP dependent unwinding disrupts hydrogen bonds between strands of DNA Requires ssDNA site to start In prokaryotes has primase activity i e puts down small section of RNA as DNA primer 3 SSB single stranded DNA binding protein Holds unwound strands open and prevents them from reannealing 4 Polymerase I and III Make the DNA many subunits with multiple domains All DNA polymerase have similar properties whether Prok or Euk 1 Have an active site that selects complementary bases by Watson and Crick interactions with the template strand 2 Synthesize DNA in 5 3 direction and anti parallel to the template strand 3 Require a primer oligo with a free 3 OH to build upon Pol III Two core one g complex Each core subunit dimer These make Prokaryote polymerase were numbered Roman Numerals based upon when they were discovered i e I first then II etc o I and III are the primary ones involved in replication others in DNA repair o Pol I can proceed along template 20 bases before falling off o This association of Polymerases with template for X number of bases is called processivity Tells you how much DNA they can make o The shape can be described as resembling a right hand with thumb finger and palm domains The palm domain appears to function in catalyzing the transfer of phosphoryl groups in the phosphoryl transfer reaction DNA is palm when the enzyme is active This reaction is believed to be catalyzed by a two metal ion mechanism The finger domain functions to bind the nucleotide triphosphate with the template base The thumb domain plays a potential role in the processivity translocation and positioning of the DNA o Pol I has 3 active sites 5 3 polymerase activity 3 5 exonuclease activity proofreading 5 3 exonuclease activity RNA primer removal o o units dimer of aeq 2 and dimer of t subunits polymerase binds to a b up the holoenzyme Complex is responsible for assembly of Pol III onto DNA acting as a clamp loader by catalyzing the ATP dependent transfer of the b subunit to each strand of DNA template multiple times for lagging strand b subunit acts as sliding clamp to enhance processivity 4 6 Mbases before falling off Subunits serves as processivity switch which allows release of DNA on lagging strand 5 Ligase Seals nicks left from Okazaki fragments Replication on lagging strand is through trombone complex to allow 5 3 replication Lagging strand is copied discontinuously Know the general process initiation elongation termination A and B Initiation and Elongation requires unwinding of DNA for a single strand template Semiconservative strand Bidirectional commences at origins of replication Bidirectional is discontinuous 5 to 3 o Leading lagging strand DNA polymerases replicate DNA New base is Watson Crick base paired in the active site Processivity number of nucleotides that can be added before pol falls off DNA Pol III replicates E coli chromosome 3 polypeptides plus auxilliary subunits Rate 1 kbase sec and processivity the whole genome i e 4 6 mega bases DNA ligase seals nicks between Okazaki fragments DNA pol s are actually immobilized replication factories anchored to membrane or nuclear envelope C Termination o Termination of DNA