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Nucleic Acids: DNA, RNA and chemistry Andy Howard Introductory Biochemistry 7 October 2010 Biochemistry:Nucleic Acids II 10/07/2010 DNA & RNA structure & function DNA and RNA are dynamic molecules, but understanding their structural realities helps us understand how they work 10/07/2010 Biochemistry:Nucleic Acids II p. 2 of 49 What we’ll discuss DNA structure Characterizations B, A, and Z-DNA Dynamics Function RNA: structure & types DNA & RNA Hydrolysis alkaline RNA, DNA nucleases mRNA tRNA rRNA Small RNAs 10/07/2010 Biochemistry:Nucleic Acids II p. 3 of 49 DNA secondary structures If double-stranded DNA were simply a straightlegged 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 10/07/2010 Biochemistry:Nucleic Acids II p. 4 of 49 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 From Molecular perpendicular to helix axis Biology web-book 10/07/2010 Biochemistry:Nucleic Acids II p. 5 of 49 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 10/07/2010 Biochemistry:Nucleic Acids II p. 6 of 49 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 10/07/2010 Biochemistry:Nucleic Acids II p. 7 of 49 Cartoon of AT pair in B-DNA 10/07/2010 Biochemistry:Nucleic Acids II p. 8 of 49 Cartoon of CG pair in B-DNA 10/07/2010 Biochemistry:Nucleic Acids II p. 9 of 49 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 10/07/2010 Biochemistry:Nucleic Acids II p. 10 of 49 Helical twist (fig. 11.9a) Rotation about the backbone axis Successive basepairs rotated with respect to each other by ~ 32º 10/07/2010 Biochemistry:Nucleic Acids II p. 11 of 49 Propeller twist Improves overlap of hydrophobic surfaces Makes it harder for water to contact the less hydrophilic parts of the molecule 10/07/2010 Biochemistry:Nucleic Acids II p. 12 of 49 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 the 2’-OH gets in the way of B-RNA Broader 2.46 nm per full turn 11 bp to complete a turn Base-pairs are not perpendicular to helix axis: tilted 19º from perpendicular 10/07/2010 Biochemistry:Nucleic Acids II p. 13 of 49 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 10/07/2010 Biochemistry:Nucleic Acids II p. 14 of 49 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! 10/07/2010 Biochemistry:Nucleic Acids II p. 15 of 49 Summaries of A, B, Z DNA 10/07/2010 Biochemistry:Nucleic Acids II p. 16 of 49 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 10/07/2010 Biochemistry:Nucleic Acids II p. 17 of 49 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! 10/07/2010 Biochemistry:Nucleic Acids II p. 18 of 49 Ribonucleic acid We’re done with DNA for the moment. Let’s discuss RNA. RNA is generally, but not always, singlestranded The regions where localized base-pairing occurs (local double-stranded regions) often are of functional significance 10/07/2010 Biochemistry:Nucleic Acids II p. 19 of 49 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 % %turnSize, 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 10/07/2010 Biochemistry:Nucleic Acids II p. 20 of 49 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! 10/07/2010 Biochemistry:Nucleic Acids II p. 21 of 49 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 10/07/2010 Biochemistry:Nucleic Acids II p. 22 of 49 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! 10/07/2010 Biochemistry:Nucleic Acids II p. 23 of 49 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 10/07/2010 Biochemistry:Nucleic Acids II p. 24 of 49 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 10/07/2010 Biochemistry:Nucleic Acids II p. 25 of 49 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 10/07/2010 Biochemistry:Nucleic Acids II Phe tRNA PDB 1EVV 76 bases yeast p. 26 of 49 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 10/07/2010 Biochemistry:Nucleic Acids II p. 27 of 49 tRNA structure: overview 10/07/2010 Biochemistry:Nucleic Acids II p. 28 of 49 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. 10/07/2010 Biochemistry:Nucleic Acids II p. 29 of 49 Yeast phetRNA Note nonstandard bases and cloverleaf structure 10/07/2010 Biochemistry:Nucleic Acids II p. 30 of 49 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 10/07/2010 Biochemistry:Nucleic Acids II 23S rRNA PDB 1FFZ 602 bases Haloarcula marismortui p. 31 of 49 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 Protein Prp31 Some are catalytic complexed to U4 Typically 20-1000 bases snRNA Not terribly plentiful: ~2 % of total RNA PDB 2OZB 33 bases + 85kDa heterotetramer Human 10/07/2010 Biochemistry:Nucleic Acids II p. 32 of 49 iClicker quiz 1. Shown is the lactim form of which nucleic acid base? Uracil Guanine Adenine Thymine None of the above HN O 10/07/2010 Biochemistry:Nucleic Acids II N OH lactim p. 33 of 49 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 10/07/2010 Biochemistry:Nucleic Acids II p. 34 of 49 Unusual bases in RNA mRNA, sRNA mostly ACGU rRNA, tRNA have some odd ones 10/07/2010 Biochemistry:Nucleic Acids II p. 35 of 49 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 QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. snoRNA77 courtesy Wikipedia 10/07/2010 Biochemistry:Nucleic Acids II p. 36 of 49 siRNAs and gene silencing QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. 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 10/07/2010 Biochemistry:Nucleic Acids II Viral p19 protein complexed to human 19-base siRNA PDB 1R9F 1.95Å 17kDa protein p. 37 of 49 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 10/07/2010 Biochemistry:Nucleic Acids II p. 38 of 49 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 10/07/2010 Biochemistry:Nucleic Acids II p. 39 of 49 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 10/07/2010 Biochemistry:Nucleic Acids II p. 40 of 49 Why alkaline hydrolysis works Cyclic phosphate intermediate stabilizes cleavage product 10/07/2010 Biochemistry:Nucleic Acids II p. 41 of 49 The cyclic intermediate Hydroxyl or water can attack fivemembered Pcontaining ring on either side and leave the –OP on 2’ or on 3’. O H N O O O- O P N 10/07/2010 Biochemistry:Nucleic Acids II O OO O P O- O p. 42 of 49 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! 10/07/2010 Biochemistry:Nucleic Acids II p. 43 of 49 Enzymatic cleavage of oligoand 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 10/07/2010 Biochemistry:Nucleic Acids II p. 44 of 49 An a-specific exonuclease 10/07/2010 Biochemistry:Nucleic Acids II p. 45 of 49 A b-specific exonuclease 10/07/2010 Biochemistry:Nucleic Acids II p. 46 of 49 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 10/07/2010 Biochemistry:Nucleic Acids II p. 47 of 49 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 10/07/2010 Biochemistry:Nucleic Acids II PDB 1KF8 13.6 kDa monomer bovine p. 48 of 49 Variety of nucleases 10/07/2010 Biochemistry:Nucleic Acids II p. 49 of 49