10/02/08Biochemistry: Nucleic Acid Chem&Struct

Nucleic AcidStructure

Andy HowardIntroductory Biochemistry

7 October 2008

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What we’ll discuss Small RNAs DNA & RNA

Hydrolysis RNA, DNA Restriction enzymes

DNA sequencing DNA secondary

structure: A, B, Z Folding kinetics

Supercoils Nucleosomes Chromatin and

chromosomes Lab synthesis of genes tRNA & rRNA structure

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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 basesQuickTime™ and a

TIFF (Uncompressed) decompressorare needed to see this picture.snoRNA77

courtesy 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

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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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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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. 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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Quirky math question to ponder Numbers can be palindromic: 484, 1331, 727, 595… Some numbers that are palindromic have

squares that are palindromic… 222 = 484, 1212 = 14641, . . . Question: if a number is perfect square

and a palindrome, is its square root a palindrome? (answer will be given orally)

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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: 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

QuickTime™ and aTIFF (Uncompressed) decompressor

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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

QuickTime™ and aTIFF (Uncompressed) decompressor

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HhaI methyltransferasePDB 1SVU2.66Å; 72 kDa dimer

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

Structure courtesy steve.gb.com

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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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Sanger dideoxy method Incorporates DNA replication as an analytical

tool for determining sequence Uses short primer that attaches to the 3’ end of

the ssDNA, after which a specially engineered DNA polymerase

Each vial includes one dideoxyXTP and 3 ordinary dXTPs; the dideoxyXTP will be incorporated but will halt synthesis because the 3’ position is blocked.

See figs. 11.3 & 11.4 for how these are read out

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Automating dideoxy sequencing Laser fluorescence detection allows for

primer identification in real time An automated sequencing machine can

handle 4500 bases/hour That’s one of the technologies that has

made large-scale sequencing projects like the human genome project possible

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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

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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 deep

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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 and 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

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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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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