Nucleic Acids: Chemistry & Structure

10/08/2009Biochemistry:Nucleic Acids I

Nucleic Acids:Chemistry & Structure

Andy HowardIntroductory Biochemistry

8 October 2009

10/08/2009 Biochemistry:Nucleic Acids I

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What we’ll discuss

Nucleic acid chemistry Pyrimidines: C, U, T

Purines: A, G Nucleosides & nucleotides

Oligo- and polynucleotides

DNA duplexes and helicity

DNA sequencing DNA secondary structure: A, B, Z

Folding kinetics


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Chemistry Nobel Prize 2009 Structural studies of the ribosome

Venki Ramakrishnan, LMB Cambridge Thomas Steitz, HHMI Yale University Ada Yonath, Weizmann Institute

QuickTime™ and a decompressor

are needed to see this picture.






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Pyrimidines Single-ring nucleic acid bases 6-atom ring; always two nitrogens in the ring, meta to one another

Based on pyrimidine, although pyrimidine itself is not a biologically important molecule

Variations depend on oxygens and nitrogens attached to ring carbons

Tautomerization possible Note line of symmetry in pyrimidine structure

N

N

pyrimidine

1

2

3

4

5

6


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Uracil and thymine

Uracil is a simple dioxo derivative of pyrimidine: 2,4-dioxopyrimidine

Thymine is 5-methyluracil Uracil is found in RNA; Thymine is found in DNA

We can draw other tautomers where we move the protons to the oxygens

HN

OHN O

uracil

HN

O NH

O

thymine


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Tautomers

Lactam and Lactim forms

Getting these right was essential to Watson & Crick’s development of the DNA double helical model

HN

OHN O

uracil - lactam

NH

ONO

uracil - lactimH

HN

O NH

O

thymine - lactam

HN

O N OH

thymine - lactim


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Cytosine

This is 2-oxo,4-aminopyrimidine

It’s the other pyrimidine base found in DNA & RNA

Spontaneous deamination (CU) Again, other tautomers can be drawn

N

OHN NH2

cytosine


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Cytosine:amino and imino forms Again, this tautomerization needs to be kept in mind

N

OHN NH

cytosine -imino form

N

OHN NH2

cytosine -amino form


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Purines Derivatives of purine; again, the root molecule isn’t biologically important

Six-membered ring looks a lot like pyrimidine

Numbering works somewhat differently: note that the glycosidic bonds will be to N9, whereas it’s to N1 in pyrimidines

HN

NN

N

purine

1

2

3

4

56 7

8

9


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Adenine This is 6-aminopurine Found in RNA and DNA We’ve seen how important adenosine and its derivatives are in metabolism

Tautomerization happens here too

N

N

NH2

N

HN

adenine - amino form

HN

N

NH

N

HN

adenine - imino form


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Guanine This is 2-amino-6-oxopurine Found in RNA, DNA Lactam, lactim forms

HN

NNH2N

HN

O

guanine - lactam

HN

NNH2N

N

OH

guanine - lactim


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Other natural purines

Hypoxanthine and xanthine are biosynthetic precursors of A & G

Urate is important in nitrogen excretion pathways


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Tautomerization and H-bonds Lactam forms predominate at neutral pH

This influences which bases are H-bond donors or acceptors

Amino groups in C, A, G make H-bonds So do ring nitrogens at 3 in pyrimidines and 1 in purines

… and oxygens at 4 in U,T, 2 in C, 6 in G


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Nucleosides

As mentioned in ch. 8, these are glycosides of the nucleic acid bases

Sugar is always ribose or deoxyribose

Connected nitrogen is: N1 for pyrimidines (on 6-membered ring) N9 for purines (on 5-membered ring)

NR1R2

OH

HO

O

HO

N-glycoside of ribofuranose


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Pyrimidine nucleosides

Drawn here in amino and lactam forms

OH

OHHO

ON

ONH2N

cytidine

OH

OHHO

ON

ONH

O

uridine


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Pyrimidine deoxynucleosides

OH

OHH

ON

ONH

O

2'-deoxyuridine

OH

OHH

ON

ONH

O

2'-deoxythymidineOH

OH

ON

ONH2N

deoxycytidine


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A tricky nomenclature issue Remember that thymidine and its phosphorylated derivatives ordinarily occur associated with deoxyribose, not ribose

Therefore many people leave off the deoxy- prefix in names of thymidine and its derivatives: it’s usually assumed.


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Purine nucleosides

Drawn in amino and lactam forms

OH

HO

HO

O

N

N

NH2

N

N

adenosine

OH

HO

HO

O

N

N

O

HN

H2N N

guanosine


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Purine deoxynucleosides

OH

HO

O

N

N

O

HN

H2N N

deoxyguanosine

OH

HO

O

N

N

NH2

N

N

deoxyadenosine


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Conformations around the glycosidic bond Rotation of the base around the glycosidic bond is sterically hindered

In the syn conformation there would be some interference between the base and the 2’-hydroxyl of the sugar

Therefore pyrimidines are always anti, and purines are usually anti

Furanose and base rings are roughly perpendicular


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Glycosidic bonds

This illustrates the roughly perpendicular positionings of the base and sugar rings


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Solubility of nucleosides and lability of glycosidic linkages The sugar makes nucleosides more soluble than the free bases

Nucleosides are generally stable to basic hydrolysis at the glycosidic bond

Acid hydrolysis: Purines: glycosidic bond fairly readily hydrolyzed

Pyrimidines: resistant to acid hydrolysis


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Chirality in nucleic acids Bases themselves are achiral Four asymmetric centers in ribofuranose, counting the glycosidic bond.

Three in deoxyribofuranose Glycosidic bond is one of those 4 or 3.

Same for nucleotides:phosphates don’t add asymmetries


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Mono-phosphorylated nucleosides

We have specialized names for the 5’-phospho derivatives of the nucleosides, i.e. the nucleoside monophosphates:

They are nucleotides Adenosine 5’-monophosphate = AMP = adenylate

GMP = guanylate CMP = cytidylate UMP = uridylate

P

O

O-

O-O

HO

HO

O

N

N

NH2

N

N

adenylate


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pKa’s for base N’s and PO4’sNucleotide

pKa base-N

pK1 of PO4

pK2 of PO4

5’-AMP 3.8(N-1) 0.9 6.1

5’-GMP 9.4 (N-1)

0.7 6.1

2.4 (N-7)

5’-CMP 4.5 (N-3)

0.8 6.3

5’-UMP 9.5 (N-3)

1.0 6.4


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UV absorbance These aromatic rings absorb around 260


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Deoxynucleotides Similar nomenclature dAMP = deoxyadenylate

dGMP = deoxyguanylate

dCMP = deoxycytidylate

dTTP (= TTP) = deoxythymidylate = thymidylate

P

O

O-

O-O

HO

O

N

N

O

HN

H2N N

deoxyguanylate


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Di and triphosphates Phosphoanhydride bonds link second and perhaps third phosphates to the 5’-OH on the ribose moiety

OHHO

O

N

O

N

H2NP

O

O

O-O-O

P

O

O-O

P

O

OH

cytidine triphosphate

Mg2+


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Cyclic phospho-diesters

3’ and 5’ hydroxyls are both involvedin -O-P-O bonds

cAMP and cGMP are the important ones(see earlier in the course!)


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Oligomers and Polymers

Monomers are nucleotides or deoxynucleotides

Linkages are phosphodiester linkages between 3’ of one ribose and 5’ of the next ribose

It’s logical to start from the 5’ end for synthetic reasons


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Typical DNA dinucleotide

Various notations: this is pdApdCp Leave out the p’s if there’s a lot of them!

P

O

-O O-

O

O

NN

O

HN

NH2

N P

O

-O

O

O

O

ON

O N NH2

P

O--O

O


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DNA structure

Many years of careful experimental work enabled fabrication of double-helical model of double-stranded DNA

Explained [A]=[T], [C]=[G]

Specific H-bonds stabilize double-helical structure: see fig. 10.20


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What does double-stranded DNA really look like? Picture on previous slide emphasizes only the H-bond interactions; it ignores the orientation of the sugars, which are actually tilted relative to the helix axis

Planes of the bases are almost perpendicular to the helical axes on both sides of the double helix


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Sizes (cf fig. 10.20, 11.7)

Diameter of the double helix: 2.37nm Length along one full turn:10.4 base pairs = pitch = 3.40nm

Distance between stacked base pairs = rise = 0.33 nm

Major groove is wider and shallower;minor groove is narrower and deeper


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What stabilizes this?

Variety of stabilizing interactions Stacking of base pairs Hydrogen bonding between base pairs

Hydrophobic effects (burying bases, which are less polar)

Charge-charge interactions:phosphates with Mg2+ and cationic proteins

Courtesy dnareplication.info


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How close to instability is it? Pretty close. Heating DNA makes it melt: fig. 11.14

pH > 10 separates strands too The more GC pairs, the harder it is to melt DNA thermally Weaker stacking interactions in A-T One more H-bond per GC than per AT


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iClicker quiz, 1st question

1. What positions of a pair of aromatic rings leads to stabilizing interactions? (a) Parallel to one another (b) Perpendicular to one another (c) At a 45º angle to one another (d) Both (a) and (b) (e) All three: (a), (b), and ( c)


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iClicker question 2

2. Which has the highest molecular mass among the compounds listed? (a) cytidylate (b) thymidylate (c) adenylate (d) adenosine triphosphate (e) they’re all the same MW


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Base composition for DNA

As noted, [A]=[T], [C]=[G] because of base pairing

[A]/[C] etc. not governed by base pairing Can vary considerably (table 10.3) E.coli : [A], [C] about equal Mycobacterium tuberculosis: [C] > 2*[A] Mammals: [C] < 0.74*[A]


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Supercoiling Refers to levels of organization of DNA beyond the immediate double-helix

We describe circular DNA as relaxed if the closed double helix could lie flat

It’s underwound or overwound if the ends are broken, twisted, and rejoined.

Supercoils restore 10.4 bp/turn relation upon rejoining


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Supercoiling and flat DNA

Diagram courtesy SIU Carbondale


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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


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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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Nucleic Acids: Chemistry & Structure