Nucleic Acid Chemistry & Structure

1. Nucleic AcidChemistry & Structure Andy HowardIntroductory Biochemistry2 October 2008 Biochemistry: Nucleic Acid Chem&Struct

2. What we’ll discuss • Syn, anti revisited • Nucleotides • Oligo- and polynucleotides • DNA duplexes and helicity • RNA: structure & types Biochemistry: Nucleic Acid Chem&Struct

3. Syn versus anti Biochemistry: Nucleic Acid Chem&Struct

4. Mono-phosphorylated nucleosides • We have specialized names for the 5’-phospho derivatives of the nucleosides, i.e. the nucleoside monophosphates: • They are nucleotides • Adenosine 5’-monophosphate = AMP = adenylate • GMP = guanylate • CMP = cytidylate • UMP = uridylate Biochemistry: Nucleic Acid Chem&Struct

5. pKa’s for base N’s and PO4’s Biochemistry: Nucleic Acid Chem&Struct

6. UV absorbance • These aromatic rings absorb around 260 Biochemistry: Nucleic Acid Chem&Struct

7. Deoxynucleotides • Similar nomenclature • dAMP = deoxyadenylate • dGMP = deoxyguanylate • dCMP = deoxycytidylate • dTTP (= TTP) = deoxythymidylate = thymidylate Biochemistry: Nucleic Acid Chem&Struct

8. Cyclic phospho-diesters • 3’ and 5’ hydroxyls are both involvedin -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!) Biochemistry: Nucleic Acid Chem&Struct

9. Di- and triphosphates • Phosphoanhydride bonds link second and perhaps third phosphates to the 5’-OH on the ribose moiety Biochemistry: Nucleic Acid Chem&Struct

10. 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 Biochemistry: Nucleic Acid Chem&Struct

11. 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 Biochemistry: Nucleic Acid Chem&Struct

12. 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 Biochemistry: Nucleic Acid Chem&Struct

13. 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 Biochemistry: Nucleic Acid Chem&Struct

14. Typical DNA dinucleotide • Various notations: this is pdApdCp • Leave out the p’s if there’s a lot of them! Biochemistry: Nucleic Acid Chem&Struct

15. 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 Biochemistry: Nucleic Acid Chem&Struct

16. 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 Biochemistry: Nucleic Acid Chem&Struct

17. 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 Biochemistry: Nucleic Acid Chem&Struct

18. 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 Biochemistry: Nucleic Acid Chem&Struct

19. 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 Biochemistry: Nucleic Acid Chem&Struct

20. 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) Biochemistry: Nucleic Acid Chem&Struct

21. 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 Biochemistry: Nucleic Acid Chem&Struct

22. 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] Biochemistry: Nucleic Acid Chem&Struct

23. Molar ratios for various organisms’ DNA (table 10.3) Biochemistry: Nucleic Acid Chem&Struct

24. 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! Biochemistry: Nucleic Acid Chem&Struct

25. The Watson-Crick contribution • Interpreting the X-ray fiber diffraction photographs taken by Rosalind Franklin and Maurice Wilkins, W&C built a ball-and-stick model for a two-stranded form of DNA • They were able to show that their model was consistent with Franklin’s data Biochemistry: Nucleic Acid Chem&Struct

26. 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 Biochemistry: Nucleic Acid Chem&Struct

27. Higher levels of organization • Just as with protein tertiary structure, DNA structure has higher levels beyond the base-pairing, 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 Biochemistry: Nucleic Acid Chem&Struct

28. 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 Biochemistry: Nucleic Acid Chem&Struct

29. Supercoiling and flat DNA Diagram courtesy SIU Carbondale Biochemistry: Nucleic Acid Chem&Struct

30. Ribonucleic acid • We’re done with DNA for the moment. • Let’s discuss RNA. • RNA is generally, but not always, single-stranded • The regions where localized base-pairing occurs (local double-stranded regions) often are of functional significance Biochemistry: Nucleic Acid Chem&Struct

31. 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 % %turn- Size, Hbond Role RNA over bases stabil.? in translation mRNA 3 25 50-104 no protein template tRNA 15 21 55-94 yes aa activation rRNA 80 50 102-104 yes transl. catalysis & scaffolding sRNA 2 4 12-200 yes various Biochemistry: Nucleic Acid Chem&Struct

32. Unusual bases in RNA • mRNA, sRNA mostly A,C,G,U • rRNA, tRNA have some odd ones Biochemistry: Nucleic Acid Chem&Struct

33. 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 Biochemistry: Nucleic Acid Chem&Struct

34. 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 Biochemistry: Nucleic Acid Chem&Struct

35. 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 Biochemistry: Nucleic Acid Chem&Struct

36. 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 Biochemistry: Nucleic Acid Chem&Struct

37. Prokaryotic ribosomes (fig.10.25a) Biochemistry: Nucleic Acid Chem&Struct

38. Eukaryotic ribosomes (fig. 10.25b) Biochemistry: Nucleic Acid Chem&Struct

39. Transfer RNA • 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 A/T site tRNA model based on cryoEM complex PDB 1QZA Biochemistry: Nucleic Acid Chem&Struct

40. 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 Dieter Söll Biochemistry: Nucleic Acid Chem&Struct

41. Small nuclear RNAs • snRNA found mostly in nucleus • 100-200 nucleotides • closely associated with proteins& with other RNA molecules • 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 Image courtesy Richard Lührmann, Göttingen Biochemistry: Nucleic Acid Chem&Struct

42. 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 snoRNA77courtesy Wikipedia Biochemistry: Nucleic Acid Chem&Struct

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. Biochemistry: Nucleic Acid Chem&Struct