10/07/2010Biochemistry:Nucleic Acids II
Nucleic Acids:DNA, RNA and chemistryAndy Howard
Introductory Biochemistry7 October 2010
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DNA & RNA structure & function DNA and RNA are dynamic molecules, but understanding their structural realities helps us understand how they work
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What we’ll discuss 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
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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
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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
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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
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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 deepFrom Berg et al.,Biochemistry
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Cartoon of AT pair in B-DNA
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Cartoon of CG pair in B-DNA
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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
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Helical twist (fig. 11.9a) Rotation about the backbone axis
Successive base-pairs rotated with respect to each other by ~ 32º
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Propeller twist
Improves overlap of hydrophobic surfaces
Makes it harder for water to contact the less hydrophilic parts of the molecule
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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
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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
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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!
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Summaries of A, B, Z DNA
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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
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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!
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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
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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 cellsType %%turn- Size, Partly Role
RNA over bases DS?mRNA 3 25 50-104 no protein
templatetRNA 15 21 55-90 yes aa activationrRNA 80 50 102-104 no transl.
catalysis & scaffolding
sRNA 2 4 15-103 ? various
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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!
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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
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mRNA processing in Eukaryotes
# 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!
Genomic DNA
Unmodified mRNA produced therefrom
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Splicing: quick summary
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
Genomic DNA
Unmodified mRNA produced therefrom
exon intron exon exonintron intron
exon exon exonsplicing
translation
transcription
(Mature transcript)
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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
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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
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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
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tRNA structure: overview
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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.
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Yeast phe-tRNA Note nonstandard bases and cloverleaf structure
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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
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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
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iClicker quiz 1. Shown is the lactim form of which nucleic acid base? Uracil Guanine Adenine Thymine None of the above
HN
O N OH
lactim
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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
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Unusual bases in RNA mRNA, sRNA mostly ACGU
rRNA, tRNA have some odd ones
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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 aTIFF (Uncompressed) decompressor
are needed to see this picture.snoRNA77courtesy Wikipedia
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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
QuickTime™ and aTIFF (Uncompressed) decompressor
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Viral p19 protein complexed to human 19-base siRNAPDB 1R9F1.95Å17kDa protein
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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
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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
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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
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Why alkaline hydrolysis works Cyclic phosphate intermediate stabilizes cleavage product
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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’.
P
O
O-
O-
O
OO
ON
OHN
O
P
O
O-
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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!
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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
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An a-specific exonuclease
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A b-specific exonuclease
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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
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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 monomerbovine
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Variety of nucleases
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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
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What do we mean by palindromic?
In ordinary language, it means a phrase that reads the same forward and back: Madam, I’m Adam. (Genesis 3:20) Eve, man, am Eve. Sex at noon taxes. Able was I ere I saw Elba. (Napoleon) A man, a plan, a canal: Panama! (T. Roosevelt)
With DNA it means the double-stranded sequence is identical on both strands
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Palindromic DNA G-A-A-T-T-C Single strand isn’t symmetric: but the combination with the complementary strand is:
G-A-A-T-T-CC-T-T-A-A-G
These kinds of sequences are the recognition sites for restriction endonucleases. This particular hexanucleotide is the recognition sequence for EcoRI.
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Cleavage by restriction endonucleases
Breaks can be cohesive (if they’re off-center within the sequence) or
non-cohesive (blunt) (if they’re at the center) EcoRI leaves staggered 5’-termini: cleaves between initial G and A
PstI cleaves CTGCAG between A and G, so it leaves staggered 3’-termini
BalI cleaves TGGCCA in the middle: blunt!
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iClicker question 3:
3. Which of the following is a potential restriction site? (a) ACTTCA (b) AGCGCT (c) TGGCCT (d) AACCGG (e) none of the above.
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Example for EcoRI 5’-N-N-N-N-G-A-A-T-T-C-N-N-N-N-3’3’-N-N-N-N-C-T-T-A-A-G-N-N-N-N-5’
Cleaves G-A on top, A-G on bottom: 5’-N-N-N-N-GA-A-T-T-C-N-N-N-N-3’3’-N-N-N-N-C-T-T-A-AG-N-N-N-N-5’
Protruding 5’ ends:5’-N-N-N-N-G A-A-T-T-C-N-N-N-N-3’3’-N-N-N-N-C-T-T-A-A G-N-N-N-N-5’
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How often? 4 types of bases So a recognition site that is 4 bases long will occur once every 44 = 256 bases on either strand, on average
6-base site: every 46= 4096 bases, which is roughly one gene’s worth
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EcoRI structure
Dimeric structure enables recognition of palindromic sequence
sandwich in each monomer
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EcoRI pre-recognition complexPDB 1CL857 kDa dimer + DNA
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Methylases A typical bacterium protects its own DNA against cleavage by its restriction endonucleases by methylating a base in the restriction site
Methylating agent is generally S-adenosylmethionine
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HhaI methyltransferasePDB 1SVU2.66Å; 72 kDa dimer
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Structure courtesy steve.gb.com
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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
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How this 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
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Use of restriction enzymes
Nature made these to protect bacteria; we use them to cleave DNA in analyzable ways Similar to proteolytic digestion of proteins
Having a variety of nucleases means we can get fragments in multiple ways
We can amplify our DNA first Can also be used in synthesis of inserts that we can incorporate into plasmids that enable us to make appropriate DNA molecules in bacteria
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Intercalating agents
Generally: aromatic compounds that can form -stack interactions with bases
Bases must be forced apart to fit them in
Results in an almost ladderlike structure for the sugar-phosphate backbone locally
Conclusion: it must be easy to do local unwinding to get those in!
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Instances of inter-calators
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Denaturing and Renaturing DNA
See Figure 11.17 When DNA is heated to 80+ degrees Celsius, its UV absorbance increases by 30-40%
This hyperchromic shift reflects the unwinding of the DNA double helix
Stacked base pairs in native DNA absorb less light
When T is lowered, the absorbance drops, reflecting the re-establishment of stacking
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Heat denaturation Figure 11.14
Heat denaturation of DNA from various sources, so-called melting curves. The midpoint of the melting curve is defined as the melting temperature, Tm.(From Marmur, J., 1959. Nature 183:1427–1429.)
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GC content vs. melting temp High salt and no chelators raises the melting temperature
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How else can we melt DNA? High pH deprotonates the bases so the H-bonds disappear
Low pH hyper-protonates the bases so the H-bonds disappear
Alkalai is better: it doesn’t break the glycosidic linkages
Urea, formamide make better H-bonds than the DNA itself so they denature DNA
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What happens if we separate the strands?
We can renature the DNA into a double helix
Requires re-association of 2 strands: reannealing
The realignment can go wrong Association is 2nd-order, zippering is first order and therefore faster
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Steps in denaturation and renaturation
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Rate depends on complexity The more complex DNA is, the longer it takes for nucleation of renaturation to occur
“Complex” can mean “large”, but complexity is influenced by sequence randomness: poly(AT) is faster than a random sequence
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Second-order kinetics Rate of association: -dc/dt = k2c2
Boundary condition is fully denatured concentration c0 at time t=0:
c / c0 = (1+k2c0t)-1
Half time is t1/2 = (k2c0)-1
Routine depiction: plot c0t vs. fraction reassociated (c /c0) and find the halfway point.
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Typical c0t curves
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Hybrid duplexes We can associate
DNA from 2 species Closer relatives hybridize better
Can be probed one gene at a time
DNA-RNA hybrids can be used to fish out appropriate RNA molecules
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GC-rich DNA is denser
DNA is denser than RNA or protein, period, because it can coil up so compactly
Therefore density-gradient centrifugation separates DNA from other cellular macromolecules
GC-rich DNA is 3% denser than AT-rich
Can be used as a quick measure of GC content
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Density as
function of GC content