BCH 380 1st editionFinal Exam Study GuideLecture 33 (April 20) Nucleic AcidsKnow nomenclature for base, nucleoside, nucleotide, oligonucleotideNucleosides:o Nucleic acid base plus sugar- sugar is pentoseo Ribose or deoxyribose Ribonucleoside or deoxyribonucleosideo Base is linked to sugar by N-glycosidic bond versus O-glycosidic bond in carbohydrates- Glycosidic bond is always betao Nucleosides are named by adding –idine to pyrimidine names and –osine to purine namesSugar numbering uses the [‘] to distinguish from base numberingNote beta conformation about anomeric carbonRibonucleosidesPurines: Guanosine, AdenosinePyrimidines: Ribothymidine (?), Cytidine, UridineDeoxyribonucleosidesPurines: 2’-deoxguanosine, 2’-deoxyadenosinePyrimidines: Thymidine (?), 2’-deoxycytidine, 2’-deoxyuridineNucleotides:• Nucleotide is Sugar + Base + Phosphate• Naming can be written out or annotated5’-triphosphate-2’-deoxyguanosine: 5’-dGTPUridine-3’-monophosphate: 3’-rUMP3’,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 carrierGTP: energy for protein synthesisCTP: energy for phospholipid synthesis UTP: energy for carbohydrate synthesisAlmost always bound to Mg+- O2 of pyrimidines over plane of sugar in synH-8 of purine over plane of sugar in antiBasic 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….etco Oligonucleotide (>10 nt) to polynucleotide - The polymer forms 3’-5’ with release of pyrophosphate from incoming NTP and phosphodiester bond formationThis 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 Ko They form an octamer “core” For DNA to wrap around- 3 Types of RNAo mRNA- messenger RNAo tRNA- transfer amino acidso 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 pairingLecture 34 (April 22) DNA ReplicationWhat 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 replication1. 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 ss-DNA 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 re-annealing.4. Polymerase I and III: Make the DNA, many subunits with multiple domains. All DNA polymerase have similar properties whether Prok or Euk1. 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.- 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 youhow much DNA they can make.o The shape can be described as resembling a righthand with thumb, finger, and palm domains. Thepalm domain appears to function in catalyzing thetransfer of phosphoryl groups in the phosphoryltransfer reaction. DNA is palm when the enzymeis active. This reaction is believed to be catalyzedby 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 sites5’-3’ polymerase activity3’-5’ exonuclease activity (proofreading)5’-3’ exonuclease activity (RNA primer removal)ooPol III: Two core units (dimer) of (aeq)2 , one g complex and dimer of t subunits. Each core polymerase binds to a b-subunit dimerThese make 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 fallingoff. Subunits serves as processivity switch which allows release of DNA on lagging strand5. Ligase: Seals nicks left from Okazaki fragments.Replication on lagging strand is through “trombone complex” to allow 5’-3’ replicationLagging strand is copied discontinuouslyKnow the general process: initiation, elongation; termination. A and B. Initiation and Elongation- requires unwinding of DNA for a single strand templateSemiconservative strandBidirectional- commences at origins of replicationBidirectional is discontinuous- 5’to 3’ o Leading/lagging strandDNA polymerases replicate DNA. New base is Watson-Crickbase-paired in the active site. “Processivity” = number ofnucleotides that can be addedbefore pol falls off. DNA Pol III replicates