Nucleic Acids: DNA, RNA and chemistry

1. Nucleic Acids:DNA, RNA and chemistry Andy HowardIntroductory Biochemistry7 October 2010 Biochemistry:Nucleic Acids II

2. DNA & RNA structure & function • DNA and RNA are dynamic molecules, but understanding their structural realities helps us understand how they work Biochemistry:Nucleic Acids II

3. DNA structure Characterizations B, A, and Z-DNA Dynamics Function RNA:structure & types mRNA tRNA rRNA Small RNAs DNA & RNA Hydrolysis alkaline RNA, DNA nucleases Restriction enzymes DNA & RNA dynamics and density measurements What we’ll discuss Biochemistry:Nucleic Acids II

4. DNA secondary structures • If double-stranded DNA were simply a straight-legged ladder: • Base pairs would be 0.6 nm apart • Watson-Crick base-pairs have very uniform dimensions because the H-bonds are fixed lengths • But water could get to the apolar bases • So, in fact, the ladder gets twisted into a helix. • The most common helix is B-DNA, but there are others. B-DNA’s properties include: • Sugar-sugar distance is still 0.6 nm • Helix repeats itself every 3.4 nm, i.e. 10 bp Biochemistry:Nucleic Acids II

5. Properties of B-DNA • Spacing between base-pairs along helix axis = 0.34 nm • 10 base-pairs per full turn • So: 3.4 nm per full turn is pitch length • Major and minor grooves, as discussed earlier • Base-pair plane is almost perpendicular to helix axis From Molecular Biology web-book Biochemistry:Nucleic Acids II

6. Major groove in B-DNA • H-bond between adenine NH2 and thymine ring C=O • H-bond between cytosine amine and guanine ring C=O • Wide, not very deep Biochemistry:Nucleic Acids II

7. Minor groove in B-DNA • H-bond between adenine ring N and thymine ring NH • H-bond between guanine amine and cytosine ring C=O • Narrow but deep From Berg et al.,Biochemistry Biochemistry:Nucleic Acids II

8. Cartoon of AT pair in B-DNA Biochemistry:Nucleic Acids II

9. Cartoon of CG pair in B-DNA Biochemistry:Nucleic Acids II

10. What holds duplex B-DNA together? • H-bonds (but just barely) • Electrostatics: Mg2+ –PO4-2 • van der Waals interactions • - interactions in bases • Solvent exclusion • Recognize role of grooves in defining DNA-protein interactions Biochemistry:Nucleic Acids II

11. Helical twist (fig. 11.9a) • Rotation about the backbone axis • Successive base-pairs rotated with respect to each other by ~ 32º Biochemistry:Nucleic Acids II

12. Propeller twist • Improves overlap of hydrophobic surfaces • Makes it harder for water to contact the less hydrophilic parts of the molecule Biochemistry:Nucleic Acids II

13. A-DNA (figs. 11.10) • In low humidity this forms naturally • Not likely in cellular duplex DNA,but it does form in duplex RNA & DNA-RNA hybrids because the2’-OH gets in the way of B-RNA • Broader • 2.46 nm per full turn • 11 bp to complete a turn • Base-pairs are notperpendicular to helix axis:tilted 19º from perpendicular Biochemistry:Nucleic Acids II

14. Z-DNA (figs.11.10) • Forms in alternating Py-Pu sequences and occasionally in PyPuPuPyPyPu, especially if C’s are methylated • Left-handed helix rather than right • Bases zigzag across the groove Biochemistry:Nucleic Acids II

15. Getting from B to Z • Can be accomplished without breaking bonds • … even though purines have their glycosidic bonds flipped (anti -> syn) and the pyrimidines are flipped altogether! Biochemistry:Nucleic Acids II

16. Summaries of A, B, Z DNA Biochemistry:Nucleic Acids II

17. DNA is dynamic • Don’t think of these diagrams as static • The H-bonds stretch and the torsions allow some rotations, so the ropes can form roughly spherical shapes when not constrained by histones • Shape is sequence-dependent, which influences protein-DNA interactions Biochemistry:Nucleic Acids II

18. What does DNA do? • Serve as the storehouse and the propagator of genetic information:That means that it’s made up of genes • Some code for mRNAs that code for protein • Others code for other types of RNA • Genes contain non-coding segments (introns) • But it also contains stretches that are not parts of genes at all and are serving controlling or structural roles • Avoid the term junk DNA! Biochemistry:Nucleic Acids II

19. 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 Acids II

20. 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, Partly Role RNA over bases DS? mRNA 3 25 50-104 no protein template tRNA 15 21 55-90 yes aa activation rRNA 80 50 102-104 no transl. catalysis & scaffolding sRNA 2 4 15-103 ? various Biochemistry:Nucleic Acids II

21. Messenger RNA • mRNA: transcription vehicleDNA 5’-dAdCdCdGdTdAdTdG-3’RNA 3’- U G G C A U A C-5’ • typical protein is ~500 amino acids;3 mRNA bases/aa: 1500 bases (after splicing) • Additional noncoding regions (see later) brings it up to ~4000 bases = 4000*300Da/base=1,200,000 Da • Only about 3% of cellular RNA but instable! Biochemistry:Nucleic Acids II

22. Relative quantities • Note that we said there wasn’t much mRNA around at any given moment • The amount synthesized is much greater because it has a much shorter lifetime than the others • Ribonucleases act more avidly on it • We need a mechanism for eliminating it because the cell wants to control concentrations of specific proteins Biochemistry:Nucleic Acids II

23. mRNA processing in Eukaryotes Genomic DNA Unmodified mRNA produced therefrom • # bases (unmodified mRNA) = # base-pairs of DNA in the gene…because that’s how transcription works • BUT the number of bases in the unmodified mRNA > # bases in the final mRNA that actually codes for a protein • SO there needs to be a process for getting rid of the unwanted bases in the mRNA: that’s what splicing is! Biochemistry:Nucleic Acids II

24. Splicing: quick summary Genomic DNA transcription Unmodified mRNA produced therefrom exon intron exon intron exon intron • Typically the initial eukaryotic message contains roughly twice as many bases as the final processed message • Spliceosome is the nuclear machine (snRNAs + protein) in which the introns are removed and the exons are spliced together splicing exon exon exon translation (Mature transcript) Biochemistry:Nucleic Acids II

25. Heterogeneity via spliceosomal flexibility • Specific RNA sequences in the initial mRNA signal where to start and stop each intron, but with some flexibility • That flexibility enables a single gene to code for multiple mature RNAs and therefore multiple proteins Biochemistry:Nucleic Acids II

26. Transfer RNA • tRNA: tool for engineering protein synthesis at the ribosome • Each type of amino acid has its own tRNA, responsible for positioning the correct aa into the growing protein • Roughly T-shaped or Y-shaped molecules; generally 55-90 bases long • 15% of cellular RNA Phe tRNAPDB 1EVV76 basesyeast Biochemistry:Nucleic Acids II

27. Secondary and Tertiary Structure of tRNA • Extensive H-bonding creates four double helical domains, three capped by loops, one by a stem • Only one tRNA structure (alone) is known • Phenylalanine tRNA is "L-shaped" • Many non-canonical bases found in tRNA Biochemistry:Nucleic Acids II

28. tRNA structure: overview Biochemistry:Nucleic Acids II

29. Amino acid linkage to acceptor stem Amino acids are linked to the 3'-OH end of tRNA molecules by an ester bond formed between the carboxyl group of the amino acid and the 3'-OH of the terminal ribose of the tRNA. Biochemistry:Nucleic Acids II

30. Yeast phe-tRNA • Note nonstandard bases and cloverleaf structure Biochemistry:Nucleic Acids II

31. Ribosomal RNA • rRNA: catalyic and scaffolding functions within the ribosome • Responsible for ligation of new amino acid (carried by tRNA) onto growing protein chain • Can be large: mostly 500-3000 bases • a few are smaller (150 bases) • Very abundant: 80% of cellular RNA • Relatively slow turnover 23S rRNAPDB 1FFZ602 basesHaloarcula marismortui Biochemistry:Nucleic Acids II

32. Small RNA • sRNA: few bases / molecule • often found in nucleus; thus it’s often called small nuclear RNA, snRNA • Involved in various functions, including processing of mRNA in the spliceosome • Some are catalytic • Typically 20-1000 bases • Not terribly plentiful: ~2 % of total RNA Protein Prp31complexed to U4 snRNAPDB 2OZB33 bases + 85kDa heterotetramerHuman Biochemistry:Nucleic Acids II

33. iClicker quiz • 1. Shown is the lactim form of which nucleic acid base? • Uracil • Guanine • Adenine • Thymine • None of the above Biochemistry:Nucleic Acids II

34. iClicker quiz #2 • Suppose someone reports that he has characterized the genomic DNA of an organism as having 29% A and 22% T. How would you respond? • (a) That’s a reasonable result • (b) This result is unlikely because [A] ~ [T] in duplex DNA • (c) That’s plausible if it’s a bacterium, but not if it’s a eukaryote • (d) none of the above Biochemistry:Nucleic Acids II

35. Unusual bases in RNA • mRNA, sRNA mostly ACGU • rRNA, tRNA have some odd ones Biochemistry:Nucleic Acids II

36. 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 Acids II

37. siRNAs and gene silencing • Small interfering RNAs block specific protein production by base-pairing to complementary seqs of mRNA to form dsRNA • DS regions get degraded & removed • This is a form of gene silencing or RNA interference • RNAi also changes chromatin structure and has long-range influences on expression Viral p19 protein complexed to human 19-base siRNA PDB 1R9F1.95Å 17kDa protein Biochemistry:Nucleic Acids II

38. Do the differences between RNA and DNA matter? Yes! • DNA has deoxythymidine, RNA has uridine: • cytidine spontaneously degrades to uridine • dC spontaneously degrades to dU • The only dU found in DNA is there because of degradation: dT goes with dA • So when a cell finds dU in its DNA, it knows it should replace it with dC or else synthesize dG opposite the dU instead of dA Biochemistry:Nucleic Acids II

39. Ribose vs. deoxyribose • Presence of -OH on 2’ position makes the 3’ position in RNA more susceptible to nonenzymatic cleavage than the 3’ in DNA • The ribose vs. deoxyribose distinction also influences enzymatic degradation of nucleic acids • I can carry DNA in my shirt pocket, but not RNA Biochemistry:Nucleic Acids II

40. Backbone hydrolysis of nucleic acids in base(fig. 10.29) • Nonenzymatic hydrolysis in base occurs with RNA but not DNA, as just mentioned • Reason: in base, RNA can form a specific 5-membered cyclic structure involving both 3’ and 2’ oxygens • When this reopens, the backbone is cleaved and you’re left with a mixture of 2’- and 3’-NMPs Biochemistry:Nucleic Acids II

41. Why alkaline hydrolysis works • Cyclic phosphate intermediate stabilizes cleavage product Biochemistry:Nucleic Acids II

42. The cyclic intermediate • Hydroxyl or water can attack five-membered P-containing ring on either side and leave the –OP on 2’ or on 3’. Biochemistry:Nucleic Acids II

43. Consequences • So RNA is considerably less stable compared to DNA, owing to the formation of this cyclic phosphate intermediate • DNA can’t form this because it doesn’t have a 2’ hydroxyl • In fact, deoxyribose has no free hydroxyls! Biochemistry:Nucleic Acids II

44. Enzymatic cleavage of oligo- and polynucleotides • Enzymes are phosphodiesterases • Could happen on either side of the P • 3’ cleavage is a-site; 5’ is b-site. • Endonucleases cleave somewhere on the interior of an oligo- or polynucleotide • Exonucleases cleave off the terminal nucleotide Biochemistry:Nucleic Acids II

45. An a-specific exonuclease Biochemistry:Nucleic Acids II

46. A b-specific exonuclease Biochemistry:Nucleic Acids II

47. Specificity in nucleases • Some cleave only RNA, others only DNA, some both • Often a preference for a specific base or even a particular 4-8 nucleotide sequence (restriction endonucleases) • These can be used as lab tools, but they evolved for internal reasons Biochemistry:Nucleic Acids II

48. Enzymatic RNA hydrolysis • Ribonucleases operate through a similar 5-membered ring intermediate: see fig. 19.29 for bovine RNAse A: • His-119 donates proton to 3’-OP • His-12 accepts proton from 2’-OH • Cyclic intermediate forms with cleavage below the phosphate • Ring collapses, His-12 returns proton to 2’-OH, bases restored PDB 1KF813.6 kDa monomer bovine Biochemistry:Nucleic Acids II

49. Variety of nucleases Biochemistry:Nucleic Acids II

50. Restriction endonucleases • Evolve in bacteria as antiviral tools • “Restriction” because they restrict the incorporation of foreign DNA into the bacterial chromosome • Recognize and bind to specific palindromic DNA sequences and cleave them • Self-cleavage avoided by methylation • Types I, II, III: II is most important • I and III have inherent methylase activity; II has methylase activity in an attendant enzyme Biochemistry:Nucleic Acids II