Nucleic Acids: DNA, RNA and chemistry

10/07/2010Biochemistry:Nucleic Acids II

Nucleic Acids:DNA, RNA and chemistryAndy Howard

Introductory Biochemistry7 October 2010

10/07/2010 Biochemistry:Nucleic Acids II

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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

http://www.web-books.com/MoBio/

http://www.web-books.com/MoBio/


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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


are needed to see this picture.

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



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



HhaI methyltransferasePDB 1SVU2.66Å; 72 kDa dimer



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

Nucleic Acids: DNA, RNA and chemistry

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