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Learning Objectives: Nucleosides & Nucleotides Reading Handout of Chapter 2: Bases, Nucleosides, and Nucleotides Chapters 2, 3, 4 from Neidle Be able to draw all 5 nucleotides with correct numbering (G, A, T, U, C) Know the difference between nucleoside and nucleotide Know ribose and deoxyribose numbering Recognize methylation as a major modification Know diff erent sugar puckers A-form usua lly 3'-endo, 2'-exo B-form usua lly 3'-exo, 2'-endo How does sugar pucker change distance between phosphates? Know diff erence between syn and anti - when do you see syn? Know all the hydrogen bond donors and acceptors in the Watson-Crick base pairs How do donors and acceptors change when the enol form of the bases are found vs. the keto form? What base groups are found in the major and mi nor grooves? Be able to draw the Watson-Crick base pairs Understand Hoogsten pairing and what face of the bases are used for this type of bonding Define pKa and if I give you the pKas for a nucleoside you can raw the protonation states Understand the importance of stacking to duplex stabili ty A-DNA vs. B-DNA vs. Z-DNA Know bp/turn, if each is right- or left-handed, sugar pucker, syn or anti configura tion of gly cosyl bond, groove widths and depths Know the base movements - propell er, twist, roll, til t Recognize a pali ndrome sequence - be able to postulate a crucif orm structure Recognize a mi rror repeat - be able to postulate a hairpin structure Know how G quartets are stabili zed and that they inv olve 4 G residues Be able to recognize a sequence's abili ty to form H-DNA or a third strand that can bind by Hoogsteen pairing to form a triplex Nucleosides & Nucleotides Nucleotides: 1. Nitrogenous base 2. Pentose 3. Phosphate Nucleosides: 1. Nitrogenous base 2. Pentose Nucleotides & Nucleic Acids Nitrogenous bases are either pyrimidines or purines Nucleotides & Nucleic Acids Nitrogenous bases are either pyrimidines or purines Properties of Nucleotides/Bases Compound Uracil Cytosine Thymine Adenine Guanine Deoxycytidine 5’-phosphate Thymidine 5’-phosphate Deoxyadenosine 5’-phosphate Deoxyguanosine 5’-phosphate Mol. Weight 112.09 111.10 126.11 135.11 151.15 321.24 322.21 331.22 347.23 Melt Pt (˚C) 338 320-5 326 360-5 365 187 142 360 (salt) Naturally Occurring Modified Bases in tRNA Adenine 1-methyladenosine N6-methyladenosine Inosine (deaminated adenosine) 1-methylinosine Guanine 1-methylguanosine N2-methylguanosine N2, N2 -dimethylguanosine 7-methylguanosine Cytosine 3-methylcytidine 5-methylcytidine 2-thiocytidine N4-acetylcytidine Uracil Ribosylthymine (5-methyluridine) 5-methoxyuridine 5,6-dihydrouridine 4-thiouridine 5-methyl-2-thiouridine Pseudouridine (uracil attached to ribose at C5) DNA Methylation Natural DNA contains: 5-methylcytosine N6-methyladenine N4-methylcytosine 5-hydroxymethyluracil Sequence-specific methylation after DNA synthesis 0% to 100% methylation 5-methylcytosine (favors Z-DNA in CG sequences) Bacteria: methylation distinguishes self from foreign DNA (methylation after synthesis) base-base mismatch repair (nonmethylated strand scanned) Higher Eukaryotes: 5-methylcytosine is only methylated form found predominately at CpG role in gene expression (undermethylated increase in transcription) Nucleotides & Nucleic Acids Ribose sugars can be either 2’-deoxy (DNA) Nucleotides & Nucleic Acids Ribose sugars ... or have a 2’-OH (RNA) TAUTOMERS H H H H H HOOGSTEEN Nucleotides & Nucleic Acids Nucleotides & Nucleic Acids Hydrogen bonding patterns in RNA and DNA Involve ring N, carbonyls, amino groups Permits complementary association of 2 strands of nucleic acid (structure of DNA by Watson & Crick) Uridine (RNA) Nucleotides & Nucleic Acids Chargaff’s rules 1940s 1. Base composition of DNA varies from one species to another 2. DNA from different tissues of same species have same base composition 3. Base composition of DNA in given species does not change with age, nutritional state, environment 4. In all cellular DNAs, regardless of species, the number of adenosine residues is equal to the number of thymidine residues (A=T), and the number of guanosines = cytidines (G=C) A + G (purines) = C + T (pyrimidines) pKa values in nucleosides/tides Base (site of protonation) Adenine (N1) Cytosine (N3) Guanine (N7) Guanine (N1) Thymine (N3) Uracil (N3) Nucleoside 3.52 4.17 3.3 9.42 9.93 9.38 3’-Nucleotide 5’-Nucleotide 3.70 4.43 (3.5) 9.84 9.96 3.88 4.56 (3.6) 10.00 10.47 10.06 Data relate to 20 ˚C and zero salt concentration They correspond to loss of a proton for pKa > 9 and capture of a proton for pKa < 5 Nucleotides & Nucleic Acids Properties of RNA and DNA Bases hydrophobic at neutral pH, hydrophobic stacking interactions Stabilize 3D structure of nucleic acids Nucleotides & Nucleic Acids Properties of RNA and DNA Nitrogenous rings are mostly planar Resonance in cyclic rings allow nucleotide bases to absorb UV light