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Nucleic Acid Chemistry & Structure Andy Howard Introductory Biochemistry 2 October 2008 Biochemistry: Nucleic Acid Chem&Struct 10/02/08 What we’ll discuss Syn, anti revisited Nucleotides Oligo- and polynucleotides DNA duplexes and helicity RNA: structure & types 10/02/08 Biochemistry: Nucleic Acid Chem&Struct p. 2 of 43 Syn versus anti 10/02/08 Biochemistry: Nucleic Acid Chem&Struct p. 3 of 43 Monophosphorylated nucleosides NH2 N N N We have specialized names for HO the 5’-phospho derivatives of the nucleosides, i.e. the nucleoside monophosphates: They are nucleotides N O OO OP HO O adenylate Adenosine 5’-monophosphate = AMP = adenylate GMP = guanylate CMP = cytidylate UMP = uridylate 10/02/08 Biochemistry: Nucleic Acid Chem&Struct p. 4 of 43 pKa’s for base N’s and PO4’s Nucleotide pKa base-N pK1 of PO4 pK2 of PO4 5’-AMP 3.8(N-1) 0.9 6.1 5’-GMP 9.4 (N-1) 0.7 6.1 2.4 (N-7) 5’-CMP 4.5 (N-3) 0.8 6.3 5’-UMP 9.5 (N-3) 1.0 6.4 10/02/08 Biochemistry: Nucleic Acid Chem&Struct p. 5 of 43 UV absorbance These aromatic rings absorb around 260 10/02/08 Biochemistry: Nucleic Acid Chem&Struct p. 6 of 43 Deoxynucleotides O Similar nomenclature dAMP = deoxyadenylate dGMP = deoxyguanylate dCMP = deoxycytidylate dTTP (= TTP) = deoxythymidylate = thymidylate N HN H2N N N O OO OP HO O deoxyguanylate 10/02/08 Biochemistry: Nucleic Acid Chem&Struct p. 7 of 43 Cyclic phosphodiesters 3’ and 5’ hydroxyls are both involved in -O-P-O bonds, forming a 6-membered ring (-C5’-C4’-C3’-O-P-O-) cAMP and cGMP are the important ones (see previous lecture!) 10/02/08 Biochemistry: Nucleic Acid Chem&Struct p. 8 of 43 Di- and triphosphates Phosphoanhydride bonds link second and perhaps third phosphates to the 5’-OH on the ribose moiety O N O H2N O O O P P P O N O O- O O- OH O- Mg2+ OH HO cytidine triphosphate 10/02/08 Biochemistry: Nucleic Acid Chem&Struct p. 9 of 43 These are polyprotic acids They can dissociate 3 protons (XDP) or 4 protons (XTP) from their phosphoric acid groups The ionized forms are frequently associated with divalent cations (Mg2+, Mn2+, others) The -O-P-O bonds beyond the first one are actually phosphoric anhydride linkages Phosphoanhydrides are acid-labile: quantitative liberation of Pi in 1N HCl for 7 minutes @100ºC 10/02/08 Biochemistry: Nucleic Acid Chem&Struct p. 10 of 43 NTPs: carriers of chemical energy ATP is the energy currency GTP is important in protein synthesis CTP used in phospholipid synthesis UTP forms activated intermediates with sugars (e.g. UDP-glucose) … and, of course, they’re substrates to build up RNA and DNA 10/02/08 Biochemistry: Nucleic Acid Chem&Struct p. 11 of 43 Bases are information symbols Base and sugar aren’t directly involved in metabolic roles of the XTPs But different XTPs do different things, so there are recognition components to the relevant enzymatic systems that notice whether X is A, U, C, or G Even in polynucleotides the bases play an informational role 10/02/08 Biochemistry: Nucleic Acid Chem&Struct p. 12 of 43 Oligomers and Polymers Monomers are nucleotides or deoxynucleotides Linkages are phosphodiester linkages between 3’ of one ribose and 5’ of the next ribose It’s logical to start from the 5’ end for synthetic reasons 10/02/08 Biochemistry: Nucleic Acid Chem&Struct p. 13 of 43 Typical DNA dinucleotide Various notations: this is pdApdCp Leave out the p’s if there’s a lot of them! -O OP O O O -O N O- N P O O O O N -O P O HN O NH2 O N O N 10/02/08 Biochemistry: Nucleic Acid Chem&Struct NH2 p. 14 of 43 DNA structure Many years of careful experimental work enabled fabrication of double-helical model of double-stranded DNA Explained [A]=[T], [C]=[G] Specific H-bonds stabilize double-helical structure: see fig. 10.20 10/02/08 Biochemistry: Nucleic Acid Chem&Struct p. 15 of 43 What does double-stranded DNA really look like? Picture on previous slide emphasizes only the H-bond interactions; it ignores the orientation of the sugars, which are actually tilted relative to the helix axis Planes of the bases are almost perpendicular to the helical axes on both sides of the double helix 10/02/08 Biochemistry: Nucleic Acid Chem&Struct p. 16 of 43 Sizes (cf fig. 10.20, 11.7) Diameter of the double helix: 2.37nm Length along one full turn: 10.4 base pairs = pitch = 3.40nm Distance between stacked base pairs = rise = 0.33 nm Major groove is wider and shallower; minor groove is narrower and deeper 10/02/08 Biochemistry: Nucleic Acid Chem&Struct p. 17 of 43 What stabilizes this? Variety of stabilizing interactions Stacking of base pairs Hydrogen bonding between base pairs Hydrophobic effects (burying bases, which are less polar) Charge-charge interactions: phosphates with Mg2+ and cationic proteins Courtesy dnareplication.info 10/02/08 Biochemistry: Nucleic Acid Chem&Struct p. 18 of 43 How close to instability is it? Pretty close. Heating DNA makes it melt: fig. 11.14 The more GC pairs, the harder it is to melt Weaker stacking interactions in A-T One more H-bond per GC than per AT We’ll get into DNA structure a lot more later in this lecture 10/02/08 Biochemistry: Nucleic Acid Chem&Struct p. 19 of 43 iClicker quiz 1. What positions of a pair of aromatic rings leads to stabilizing interactions? (a) Parallel to one another (b) Perpendicular to one another (c) At a 45º angle to one another (d) Both (a) and (b) (e) All three: (a), (b), and ( c) 10/02/08 Biochemistry: Nucleic Acid Chem&Struct p. 20 of 43 Second iClicker question 2. Which has the highest molecular mass among the compounds listed? (a) cytidylate (b) thymidylate (c) adenylate (d) adenosine triphosphate (e) they’re all the same MW 10/02/08 Biochemistry: Nucleic Acid Chem&Struct p. 21 of 43 Base composition for DNA As noted, [A]=[T], [C]=[G] because of base pairing [A]/[C] etc. not governed by base pairing Can vary considerably (table 10.3) E.coli : [A], [C] about equal Mycobacterium tuberculosis: [C] > 2*[A] Mammals: [C] < 0.74*[A] 10/02/08 Biochemistry: Nucleic Acid Chem&Struct p. 22 of 43 Molar ratios for various organisms’ DNA (table 10.3) Source Ox Human Hen Salmon Wheat Yeast A/G 1.29 1.56 1.45 1.43 1.22 1.67 H.influenzae 1.74 E.coli K-12 1.05 B. schatz 0.7 T/C 1.43 1.75 1.29 1.43 1.18 1.92 1.54 0.95 0.6 A/T 1.04 1.0 1.06 1.02 1.00 1.03 1.07 1.09 1.12 G/C 1.00 1.0 0.91 1.02 0.97 1.20 0.91 0.99 0.89 10/02/08 Biochemistry: Nucleic Acid Chem&Struct Pur/Pyr 1.1 1.0 0.99 1.02 0.99 1.0 1.0 1.0 1.0 p. 23 of 43 What did this mean in 1950? [A]=[T] and [C]=[G] suggested that if the molecule involved two strands, there should be complementarity between them, i.e., if there’s an A on one strand, there will be a T on the other one Unfortunately it wasn’t entirely clear that the molecule was two-stranded! 10/02/08 Biochemistry: Nucleic Acid Chem&Struct p. 24 of 43 The Watson-Crick contribution Interpreting the X-ray fiber diffraction photographs taken by Rosalind Franklin and Maurice Wilkins, W&C built a ball-andstick model for a two-stranded form of DNA They were able to show that their model was consistent with Franklin’s data 10/02/08 Biochemistry: Nucleic Acid Chem&Struct QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. p. 25 of 43 So how is DNA organized? Linear sequence is simple to describe: Two strands, each very long and containing 105 - 108 bases Each base has a complementary base on the other strand Specific hydrogen bonding patterns define the complementarity 10/02/08 Biochemistry: Nucleic Acid Chem&Struct p. 26 of 43 Higher levels of organization Just as with protein tertiary structure, DNA structure has higher levels beyond the basepairing, beginning with coiling into a double helix Eukaryotes: Organization of double helix into loop structures of ~200 base pairs coiled around a protein complex called the histone octamer Further organization of those loops into larger structures culminating in formation of chromosomes Prokaryotes: similar but simpler higher-level structures culminating in (often circular) chromosomes 10/02/08 Biochemistry: Nucleic Acid Chem&Struct p. 27 of 43 Supercoiling Refers to levels of organization of DNA beyond the immediate double-helix We describe circular DNA as relaxed if the closed double helix could lie flat It’s underwound or overwound if the ends are broken, twisted, and rejoined. Supercoils restore 10.4 bp/turn relation upon rejoining 10/02/08 Biochemistry: Nucleic Acid Chem&Struct p. 28 of 43 Supercoiling and flat DNA Diagram courtesy SIU Carbondale 10/02/08 Biochemistry: Nucleic Acid Chem&Struct p. 29 of 43 Ribonucleic acid We’re done with DNA for the moment. Let’s discuss RNA. RNA is generally, but not always, singlestranded The regions where localized base-pairing occurs (local double-stranded regions) often are of functional significance 10/02/08 Biochemistry: Nucleic Acid Chem&Struct p. 30 of 43 RNA physics & chemistry RNA molecules vary widely in size, from a few bases in length up to 10000s of bases There are several types of RNA found in cells Type % %turnRNA over mRNA 3 25 tRNA 15 21 rRNA 80 50 Size, bases 50-104 55-94 102-104 Hbond stabil.? no yes yes sRNA 12-200 yes 2 4 Role in translation protein template aa activation transl. catalysis & scaffolding various 10/02/08 Biochemistry: Nucleic Acid Chem&Struct p. 31 of 43 Unusual bases in RNA mRNA, sRNA mostly A,C,G,U rRNA, tRNA have some odd ones 10/02/08 Biochemistry: Nucleic Acid Chem&Struct p. 32 of 43 Messenger RNA Contains the codons that define protein sequence Each codon (3 bases) codes for 1 amino acid Synthesized during transcription, like all other types of RNA Relatively small % of RNA mass in the cell; but short-lived, so: Higher % of RNA synthesis devoted to mRNA 10/02/08 Biochemistry: Nucleic Acid Chem&Struct p. 33 of 43 Prokaryotic mRNA One mRNA with a single promoter will contain coding information for several proteins, i.e., 1 promoter, several genes Defined stop codons show the ribosome where to put in the breaks Translation closely coupled to transcription, unlike eukaryotic systems, where they’re separated in space & time 10/02/08 Biochemistry: Nucleic Acid Chem&Struct p. 34 of 43 Eukaryotic mRNA One mRNA per protein But the mRNA will be initially synthesized with noncoding segments (introns) interspersed between the coding segments (exons): heterogeneous nuclear RNA, hnRNA snRNPs (q.v.) in nucleus splice out the introns, tying together the exons to make the mature transcript Each mRNA will end with a poly(A) tail, added after transcription 10/02/08 Biochemistry: Nucleic Acid Chem&Struct p. 35 of 43 Ribosomes and rRNA Ribosome is 65% RNA, rest protein Lots of intrastrand H-bonds Ribosomes characterized by sedimentation coefficients E.coli: 50S piece+30S piece 70S total Eukaryotes 60S + 40S 80S total rRNA has pseudouridine, ribothymidine, methylated bases 10/02/08 Biochemistry: Nucleic Acid Chem&Struct p. 36 of 43 Prokaryotic ribosomes (fig.10.25a) 10/02/08 Biochemistry: Nucleic Acid Chem&Struct p. 37 of 43 Eukaryotic ribosomes (fig. 10.25b) 10/02/08 Biochemistry: Nucleic Acid Chem&Struct p. 38 of 43 Transfer RNA QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. Each tRNA carries a specific amino acid to the ribosomal protein synthesis machine One full set of tRNA at each cellular site of protein synthesis (cytoplasm, mitochondrion, chloroplast) These are small molecules: 55-94 bases 10/02/08 Biochemistry: Nucleic Acid Chem&Struct A/T site tRNA model based on cryoEM complex PDB 1QZA p. 39 of 43 tRNA contents Many modified bases CCA on the 3’-end is attached to the amino acid Catalytic attachment of amino acid to protein is catalyzed by an adenine in one of the 50S rRNAs QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. Quic kTime™ and a TIFF (Unc ompres sed) decompress or are needed to see this picture. Dieter Söll 10/02/08 Biochemistry: Nucleic Acid Chem&Struct p. 40 of 43 Small nuclear RNAs QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. snRNA found mostly in nucleus 100-200 nucleotides Image courtesy closely associated with proteins Richard Lührmann, & with other RNA molecules Göttingen Mostly in ribonucleoprotein particles (snRNPs), which are involved in mRNA processing, converting full-length transcript into smaller transcript in which introns have been removed, leaving only the exons 10/02/08 Biochemistry: Nucleic Acid Chem&Struct p. 41 of 43 Other small RNAs 21-28 nucleotides Target RNA or DNA through complementary base-pairing Several types, based on function: Small interfering RNAs (q.v.) microRNA: control developmental timing Small nucleolar RNA: catalysts that (among other things) create the oddball bases QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. snoRNA77 courtesy Wikipedia 10/02/08 Biochemistry: Nucleic Acid Chem&Struct p. 42 of 43 iClicker question 3 Suppose you isolate an RNA molecule that consists of 1500 bases. It is probably: (a) tRNA (b) mRNA (c) rRNA (d) either mRNA or rRNA (e) none of the above. 10/02/08 Biochemistry: Nucleic Acid Chem&Struct p. 43 of 43