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Nucleic Acid Chemistry Andy Howard Introductory Biochemistry 1 May 2008 Nucleic Acid Chemistry 01 May 2008 What we’ll discuss RNA (concluded) Chromatin Packaging of DNA Nucleosomes Histones Higher levels Bacterial packaging Nucleic Acid Chemistry Nucleases Alkaline hydrolysis RNAses Restriction Endonucleases Applications of restriction endos p.2 of 43 01 May 2008 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, RNA over by mRNA 3 25 50-104 tRNA 15 21 55-90 rRNA 80 50 102-104 sRNA 2 4 30-103 Nucleic Acid Chemistry Partly DS? no yes no ? Role protein template aa activation transl. catalysis & scaffolding various p.3 of 43 01 May 2008 Messenger RNA mRNA: transcription vehicle DNA 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! Nucleic Acid Chemistry p.4 of 43 01 May 2008 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 Nucleic Acid Chemistry p.5 of 43 Phe tRNA PDB 1EVV 76 bases yeast 01 May 2008 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 Nucleic Acid Chemistry p.6 of 43 23S rRNA PDB 1FFZ 602 bases Haloarcula marismortui 01 May 2008 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 Nucleic Acid Chemistry p.7 of 43 Protein Prp31 complexed to U4 snRNA PDB 2OZB 33 bases + 85kDa heterotetramer Human 01 May 2008 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 Chemistry p.8 of 43 01 May 2008 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 Chemistry p.9 of 43 01 May 2008 Splicing: quick summary Genomic DNA transcription Unmodified mRNA produced therefrom exon intron exon intron exon intron splicing exon exon (Mature transcript) exon translation 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 Nucleic Acid Chemistry p.10 of 43 01 May 2008 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 Chemistry p.11 of 43 01 May 2008 iClicker quiz 1. Shown is the lactim form of which nucleic acid base? Uracil Guanine Adenine Thymine None of the above Nucleic Acid Chemistry HN O N OH lactim p.12 of 43 01 May 2008 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 Nucleic Acid Chemistry p.13 of 43 01 May 2008 Chromatin Discovered long before we understood molecular biology Seen to be banded objects in nuclei of stained eukaryotic cells In resting cell it exists as long slender threads, 30 nm diameter Nucleic Acid Chemistry From answers.com p.14 of 43 01 May 2008 Squishing the DNA If the double helix were fully extended, the largest human chromosome (2.4*108bp) would be 2.4*108 *0.33nm ~ 0.8*108nm=80 mm; much bigger than the cell! So we have to coil it up a lot to make it fit. Longest chromosome is 10µm long So the packing ratio is 80mm/10µm = 8000 Nucleic Acid Chemistry p.15 of 43 01 May 2008 Nucleosomes DNA-protein complexes that hold together the DNA in coiled forms at the second- and thirdlevels of organization (first is helicity itself) The proteins involved are histones Proteins rich in basic aa’s (R,K) These interact closely with DNA to facilitate appropriate coiling Nucleic Acid Chemistry p.16 of 43 Nucleosome core particle PDB 1KX5 143 bp + 108 kDa heterooctamer Xenopus 01 May 2008 Histones Characterized as H1, H2A, H2B, H3, H4 H1 involved in higher level of organization; others in nucleosome itself All are small, K&R-rich proteins Highly conserved Nucleic Acid Chemistry p.17 of 43 01 May 2008 Categories of histones Type MW,kDa #aa’s #basic #acidic H1 21 213 65 10 H2A 14 129 30 9 H2B 13.8 125 31 10 H3 15.3 135 33 11 H4 11.3 102 27 7 Nucleic Acid Chemistry p.18 of 43 01 May 2008 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 Chemistry p.19 of 43 01 May 2008 O HN Histone deactivation ONH3+ O acylated lysine 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 acylation is a crucial element in DNA organization Nucleic Acid Chemistry p.20 of 43 01 May 2008 CoASH Histone acetylation Active histone + Acetyl CoA inactive (acetylated) histone + CoASH Without the positive charges, the affinity for DNA goes down Nucleic Acid Chemistry p.21 of 43 Histone H1 PDB 1GHC 8.3 kDa monomer Chicken Histone acetyltransferase PDB 1QSO 66 kDa tetramer yeast 01 May 2008 Histone deacetylation Type III deacetylases use a non-trivial reaction: Prot-lys-NAc + NAD+ Prot-lys-NH3+ + nicotinamide + 2’-O-acetyl-ADP-ribose Part of the NAD salvage pathway Histone/protein deacetylase + histone H4 active peptide PDB 1SZD; 34 kDa “heterodimer” yeast Nucleic Acid Chemistry p.22 of 43 01 May 2008 Nucleosome structure Core octamer is two molecules each of H2A, H2B, H3, H4 Typically wraps around ~200bp of DNA DNA between nucleosomes is ~54 bp long H1 binds to linker and to core particle; but in beads-on-a-string structure, it’s often absent Nucleic Acid Chemistry p.23 of 43 01 May 2008 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 Chemistry p.24 of 43 01 May 2008 Courtesy answers.com Next level of organization H1 interacts with DNA along linker region Individual histones spiral along to form 30 nm fiber See fig.19.25 Nucleic Acid Chemistry Courtesy Johns Hopkins Univ p.25 of 43 01 May 2008 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 Chemistry p.26 of 43 01 May 2008 What about prokaryotes? No actual histones Histone-like proteins involved Bacterial DNA attached to scaffold in large loops (~100kb) This makes a nucleoid Nucleic Acid Chemistry p.27 of 43 01 May 2008 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 Chemistry p.28 of 43 01 May 2008 O Nucleases HN O N Enzymes that hydrolyze phosphodiester bonds in nucleic acids Can clip on the 3’ end or the 5’ end of the phosphorus Can operate on DNA or RNA DNA tends to be more resistant to degradation O- O O P O O OH O O O N P -O Nucleic Acid Chemistry N O- p.29 of 43 HO NH2 OH N N 01 May 2008 Alkaline hydrolysis RNA can be readily hydrolyzed nonenzymatically, particularly at high pH DNA considerably less so RNA will be completely degraded at pH 13 (0.1N NaOH) in hours; DNA untouched This will still happen at lower pH, but much more slowly Nucleic Acid Chemistry p.30 of 43 01 May 2008 Mechanism for alkaline hydrolysis of RNA (fig. 19.28) Cyclic phosphate intermediate stabilizes cleavage product Nucleic Acid Chemistry p.31 of 43 01 May 2008 Results of creating cyclic phosphate O H N O O O- O P N Hydroxyl or water can attack five-membered P-containing ring on either side and leave the –OP on 2’ or on 3’. Nucleic Acid Chemistry O O- H O O P O H p.32 of 43 O- O- H O H O H 01 May 2008 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! Nucleic Acid Chemistry p.33 of 43 01 May 2008 Enzymatic hydrolysis of RNA 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 Nucleic Acid Chemistry p.34 of 43 PDB 1KF8 13.6 kDa monomer bovine 01 May 2008 Restriction endonucleases These are sequencespecific enzymes that cleave phosphodiester bonds in DNA Found in bacteria, which use them to cleave foreign DNA Nucleic Acid Chemistry p.35 of 43 EcoRI with DNA PDB 1ERI 61 kDa dimer + 13 bp E.coli (obviously) 01 May 2008 The biology problem How does the bacterium mark its own DNA so that it does replicate its own DNA but not the foreign DNA? Answer: by methylating specific bases in its DNA prior to replication Unmethylated DNA from foreign source gets cleaved by restriction endonuclease Only the methylated DNA survives to be replicated Most methylations are of A & G, but sometimes C gets it too Nucleic Acid Chemistry p.36 of 43 01 May 2008 How it works When an unmethylated specific sequence appears in the DNA, the enzyme cleaves it When the corresponding methylated sequence appears, it doesn’t get cleaved and remains available for replication The restriction endonucleases only bind to palindromic sequences Nucleic Acid Chemistry p.37 of 43 01 May 2008 Palindromic DNA Sequences that read the same on one strand from left to right as they do on the opposite strand reading right to left Example, found in EcoRI recognition sequence: 5’-GAATTC-3’ 3’-CTTAAG-5’ Most DNA isn’t palindromic, but palindromic sequences are common enough that we can frequently find them Nucleic Acid Chemistry p.38 of 43 01 May 2008 Nomenclature for restriction endonucleases (table 19.4) Name has three pieces: 3- or 4-character designation for organism, e.g. Eco (E.coli), Kpn (Klebsiella pneoumoniae), Bam (Bacillus amyloliquefaciens) (optional) one-character designation for strain (R, H) (e.g. R is strain R of E.coli) Roman-numeral characters for enzyme Thus :EcoRI, BamH1 Simpler: XbaI Nucleic Acid Chemistry p.39 of 43 01 May 2008 Generalizations about restriction endonucleases Each binds a specific 4-7 base-pair sequence Always recognize palindromic sequences; often local dimer within enzyme lines up on the two identical strands of DNA Cleavage site can be anywhere within the sequence Methylation site typically not on the cleaved base Nucleic Acid Chemistry p.40 of 43 01 May 2008 Cf. table 19.4! Common lab endonucleases Nuclease Source ApaI BamHI EcoRI EcoRII HinDIII HpaII Sequence Acetobacter 5’GGGCCC pasteurianus 3’CCCGGG Bacillus amilo 5’GGATCC -liquifaciens 3’CCTAGG Escherichia 5’GAA*TTC coli 3’CTT*AAG E.coli 5’CC*WGG 3’GG*WCC Haemophilus 5’A*AGCTT influenzae 3’T*TCGAA Haemophilus 5’CCGG parainflu. 3’GGCC Nucleic Acid Chemistry Cut 5’-GGGCCC C-3’ 3’-C CCGGG-5’ 5’-G GATCC-3’ 3’-CCTAG G-5’ 5’-G AATTC-3’ 3’-CTTAA G-5’ 5’CCWGG-3’ 5’-GGWCC -5’ 5’-A AGCTT-3’ 3’-TTCGA A-5’ 5’-C CGG-3’ 3’-GGC C-5’ p.41 of 43 01 May 2008 More endonucleases Nuclease Source Sequence KpnI Klebsiella 5’GGTACC pneumoniae 3’CCATGG NotI Nocardia 5’GCGGCCGC otitidis 3’CGCCGGCG PstI Providencia 5’CTGCAG stuartii 164 3’GACGTC SmaI Serratia 5’CCCGGG marescens 3’GGGCCC XbaI Xanthomonas 5’TCTAGA badrii 3’AGATCT XhoI Xanthomonas 5’CTCGAG holcicola 3’GAGCTC TaqI Thermus 5’TCGA aquaticus 3’AGCT Nucleic Acid Chemistry Cut 5’-GGTAC C-3’ 3’-C CATGG-5’ 5’-GC GGCCGC-3’ 3’-CGCCGG CG-5’ 5’-CTGCA G-3’ 3’-G ACGTC-5’ 5’-CCC GGG-3’ 3’-GGG CCC-5’ 5’-T CTAGA-3’ 3’-AGATC T-5’ 5’-C TCGAG-3’ 3’-GAGCT C-5’ 5’-T CGA-3’ 3’-AGC T-5’ p.42 of 43 01 May 2008 Applications Cleaving DNA at the restriction sites Building cleavable constructs in plasmids Recombinant DNA depends on identifying restriction sites and cleaving them Identifying mutations in a population That allows studies of genetic drift DNA fingerprinting in forensics Can be combined with PCR so the starting DNA sample can be very small Nucleic Acid Chemistry p.43 of 43 01 May 2008