1 / 75

Nucleic Acids: DNA, RNA and chemistry

Nucleic Acids: DNA, RNA and chemistry. Andy Howard Introductory Biochemistry 7 October 2010. DNA & RNA structure & function. DNA and RNA are dynamic molecules, but understanding their structural realities helps us understand how they work. DNA structure Characterizations B, A, and Z-DNA

glynis
Download Presentation

Nucleic Acids: DNA, RNA and chemistry

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  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

More Related