Nucleic Acid Structure II

1. Nucleic Acid Structure II Andy HowardIntroductory Biochemistry22 October 2014 Nucleic Acid Structure II

2. DNA structure informs its functions • We will revisit several aspects of nucleic acid structure that help us understand how it operates. Nucleic Acid Structure II

3. What we’ll discuss • RNA • Types • Functions • Splicing • DNA tertiary structure • Nucleosomes • Higher-level structures • Bacterial organization Nucleic Acid Structure II

4. 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 Nucleic Acid Structure II

5. 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 yes transl. catalysis & scaffolding sRNA 2 4 15-103 yes various Nucleic Acid Structure II

6. 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 unstable! Nucleic Acid Structure II

7. 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 Nucleic Acid Structure II

8. 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! Nucleic Acid Structure II

9. 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) Nucleic Acid Structure II

10. 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 Nucleic Acid Structure II

11. 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 Yeast Phe tRNA76 basesPDB 1EVV, 2Å Nucleic Acid Structure II

12. 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 Nucleic Acid Structure II

13. tRNA structure: overview Nucleic Acid Structure II

14. 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. Nucleic Acid Structure II

15. Yeast ala-tRNA • Note nonstandard bases and cloverleaf structure Nucleic Acid Structure II

16. 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 Haloarculamarismortui 23S rRNAPDB 1FFZ602 bases Nucleic Acid Structure II

17. 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 Nucleic Acid Structure II

18. Unusual bases in RNA • mRNA, sRNA mostly ACGU • rRNA, tRNA have some odd ones Nucleic Acid Structure II

19. 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 Nucleic Acid Structure II

20. 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 Nucleic Acid Structure II

21. Chromosome structure: levels • Each of the first 4 levels compacts DNA by a factor of 6-20; those multiply up to > 104 Nucleic Acid Structure II

22. Nucleosome Structure • Chromatin, the nucleoprotein complex, consists of histones and nonhistone chromosomal proteins • Histone octamer structure has been solved • without DNA: Moudrianakis, 1991 • with DNA by Richmond • Nonhistone proteins are regulators of gene expression Nucleic Acid Structure II

23. Histone types • H2a, H2b, H3, H4 make up core particle, associating with ~146 bp of DNA:two copies of each, so: protein octamer • All histones are KR-rich, small proteins • H1 associates with ~54bp region between two nucleosomes Nucleic Acid Structure II

24. Histones: table 11.2, plus… Nucleic Acid Structure II

25. Unfolded chromatin • Treat chromatin with low ionic strength; that disrupts higher level interactions so the individual nucleosomes are strung out relative to one another like beads on a string Image courtesy U. Maine Nucleic Acid Structure II

26. Nucleosome core particle Nucleic Acid Structure II

27. Half the core particle • Note that DNA isn’t really circular: it’s a series of straight sections followed by bends (like the Advanced Photon Source ring!) Nucleic Acid Structure II

28. Histones, continued • Individual nucleosomes attach via histone H1 to seal the ends of the turns on the core and organize 40-60bp of DNA linking consecutive nucleosomes • N-terminal tails of H3 & H4 are accessible • K, S get post-translational modifications, particularly K-acetylation Nucleic Acid Structure II

29. Histone deactivation • Histones interact with DNA via +charges on lys and arg residues. • If we neutralize those charges by acetylation, the histones don’t bind as tightly to the DNA • Carefully-timed enzymatic control of histone acetylation is a crucial element in DNA organization Nucleic Acid Structure II

30. CoASH Histone acetylation Histone H1PDB 1GHC8.3 kDa monomerChicken • Active histone + Acetyl CoA  inactive (acetylated) histone + CoASH • Without the positive charges, the affinity for DNA goes down Histone acetyltransferasePDB 1QSO66 kDatetrameryeast Nucleic Acid Structure II

31. Histone deacetylation • Type III deacetylases usea non-trivial reaction:Prot-lys-NAc + NAD+ Prot-lys-NH3+ + nicotinamide +2’-O-acetyl-ADP-ribose • Part of the NAD salvage pathway Yeast Histone/protein deacetylase +histone H4 active peptide, 34kDaPDB 1SZD, 1.5Å Nucleic Acid Structure II

32. Other histone PTM • Histones can be post-translationally modified in other ways as well • Methylation: e.g. lysines 4,27 of H3 • Phosphorylation: H2A phosphorylated at several sites near “hinge” • These are correlated with acetylation and play a role in folding and function Nucleic Acid Structure II

33. How much does this coil up? • 200 bp extended would be about 50nm • The width of the core-particle disk is 5nm • So this is a tenfold reduction • Nucleosomal organization corresponds to negative supercoiling • … so DNA ends up supercoiled when we take away the histones Nucleic Acid Structure II

34. Courtesy answers.com Next level of organization • H1 interacts with ~54 base-pairs of DNA along linker region • Individual histones spiral along to form 30 nm fiber • See fig.19.25 Courtesy Johns Hopkins Univ Nucleic Acid Structure II

35. Even higher… • The 30nm fibers are attached to an RNA-protein scaffold that holds the 30nm fibers in large loops • Typical chromosome has ~200 loops • Loops are attached to scaffold at their base • Ends can rotate so it can be supercoiled Nucleic Acid Structure II

36. What aboutprokaryotes? • No actual histones • Histone-like proteins(HLPs) involved • Bacterial DNA attached to scaffold in large loops (~100kb) • This makes a nucleoid Anabaena HU-DNA complex 33 kDa1P71, 1.9Å Nucleic Acid Structure II

37. How many loops in bacteria? • Typical bacterial genome (E.coli) has 3000 open reading frames ~ 3000 genes. • Assume 500 amino acids per protein = 1500 bases per gene (ignores transcriptional elements) • Then genome is 1500 bp/gene * 3000 genes = 4.5*106 base-pairs • That’s (4.5*106 bp)/(1*105 bp/loop) = 45 loops Nucleic Acid Structure II

38. iClicker question 1 • 1. Which of the following is a potential restriction site? • (a) ACTTCA • (b) AGCGCT • (c) TGGCCT • (d) AACCGG • (e) none of the above. Nucleic Acid Structure II

39. iClicker question 2 • 2. A DNA sample has Tm=94ºC. It is probably • (a) AT-rich • (b) CG-rich • (c) characterized in the presence of chelators • (d) measured in pure water rather than buffer • (e) either (c) or (d) Nucleic Acid Structure II

40. iClicker question 3 • 3. One step in gyrase activity depends on ATP hydrolysis. It is: • (a) association of the gyrase with the DNA loop • (b) DNA cleavage • (c) DNA re-ligation and release of DNA from gyrase • (d) all of the above Nucleic Acid Structure II

41. iClicker question 4 • 4. When DNA is compressed, which of the compression steps accomplishes the most substantial reduction in size? • (a) helix to beads-on-a-string • (b) beads-on-a-string to solenoid • (c) solenoid to loop • (d) loop to 18-loop miniband • (e) 18-loop miniband to chromosome Nucleic Acid Structure II

42. iClicker quiz, question 5 • 5. Suppose a mutation in the gene coding for histone H1 makes it fold up incorrectly. How will this mutation influence DNA organization? • (a) It will prevent formation of nucleosomes • (b) It will interfere with the beads-on-a-string organization between nucleosomes • (c) It will interfere with higher-level organization involving assembly of solenoids into loops • (d) All of the above • (e) None of the above Nucleic Acid Structure II

43. iClicker question 6 • 6. When a histone becomes acetylated, the net charge on the protein • (a) goes up • (b) goes down • (c) does not change • (d) will go up or down depending on the circumstance Nucleic Acid Structure II