10/02/08 Biochemistry: Nucleic Acid Chem&Struct Nucleic Acid Chemistry & Structure Andy Howard Introductory Biochemistry 2 October 2008

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

Nucleic AcidChemistry & Structure

Andy HowardIntroductory Biochemistry

2 October 2008

10/02/08 Biochemistry: Nucleic Acid Chem&Struct p. 2 of 43

What we’ll discuss Syn, anti revisited Nucleotides Oligo- and polynucleotides DNA duplexes and helicity RNA: structure & types


Syn versus anti


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


pKa’s for base N’s and PO4’s

Nucleotide 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


UV absorbance These aromatic rings absorb around 260


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


Cyclic phospho-diesters

3’ and 5’ hydroxyls are both involvedin -O-P-O bonds, forming a 6-membered ring (-C5’-C4’-C3’-O-P-O-)

cAMP and cGMP are the important ones(see previous lecture!)


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+


These are polyprotic acids They can dissociate 3 protons (XDP) or 4

protons (XTP) from their phosphoric acid groups

The ionized forms are frequently associated with divalent cations (Mg2+, Mn2+, others)

The -O-P-O bonds beyond the first one are actually phosphoric anhydride linkages

Phosphoanhydrides are acid-labile: quantitative liberation of Pi in 1N HCl for 7 minutes @100ºC


NTPs: carriers of chemical energy ATP is the energy currency GTP is important in protein synthesis CTP used in phospholipid synthesis UTP forms activated intermediates with

sugars (e.g. UDP-glucose) … and, of course, they’re substrates to

build up RNA and DNA


Bases are information symbols Base and sugar aren’t directly involved in

metabolic roles of the XTPs But different XTPs do different things, so

there are recognition components to the relevant enzymatic systems that notice whether X is A, U, C, or G

Even in polynucleotides the bases play an informational role


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


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


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


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


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


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


How close to instability is it? Pretty close. Heating DNA makes it melt: fig. 11.14 The more GC pairs, the harder it is to

melt Weaker stacking interactions in A-T One more H-bond per GC than per AT

We’ll get into DNA structure a lot more later in this lecture


iClicker quiz

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)


Second iClicker question

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


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]


Molar ratios for various organisms’ DNA (table 10.3)

Source A/G T/C A/T G/C Pur/Pyr

Ox 1.29 1.43 1.04 1.00 1.1Human 1.56 1.75 1.0 1.0 1.0Hen 1.45 1.29 1.06 0.91 0.99Salmon 1.43 1.43 1.02 1.02 1.02Wheat 1.22 1.18 1.00 0.97 0.99Yeast 1.67 1.92 1.03 1.20 1.0H.influenzae 1.74 1.54 1.07 0.91 1.0E.coli K-12 1.05 0.95 1.09 0.99 1.0B. schatz 0.7 0.6 1.12 0.89 1.0


What did this mean in 1950?

[A]=[T] and [C]=[G] suggested that if the molecule involved two strands, there should be complementarity between them, i.e., if there’s an A on one strand, there will be a T on the other one

Unfortunately it wasn’t entirely clear that the molecule was two-stranded!


The Watson-Crick contribution

Interpreting the X-ray fiber diffraction photographs taken by Rosalind Franklin and Maurice Wilkins, W&C built a ball-and-stick model for a two-stranded form of DNA

They were able to show that their model was consistent with Franklin’s data

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.




So how is DNA organized?

Linear sequence is simple to describe: Two strands, each very long and

containing 105 - 108 bases Each base has a complementary base on

the other strand Specific hydrogen bonding patterns

define the complementarity


Higher levels of organization Just as with protein tertiary structure, DNA

structure has higher levels beyond the base-pairing, beginning with coiling into a double helix

Eukaryotes: Organization of double helix into loop structures

of ~200 base pairs coiled around a protein complex called the histone octamer

Further organization of those loops into larger structures culminating in formation of chromosomes

Prokaryotes: similar but simpler higher-level structures culminating in (often circular) chromosomes


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


Supercoiling and flat DNA

Diagram courtesy SIU Carbondale


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


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, Hbond Role

RNA over bases stabil.? in translation

mRNA 3 25 50-104 no protein template

tRNA 15 21 55-94 yes aa activation

rRNA 80 50 102-104 yes transl. catalysis

& scaffolding

sRNA 2 4 12-200 yes various


Unusual bases in RNA mRNA, sRNA mostly A,C,G,U rRNA, tRNA have some odd ones


Messenger RNA Contains the codons that define protein

sequence Each codon (3 bases) codes for 1 amino acid Synthesized during transcription, like all other

types of RNA Relatively small % of RNA mass in the cell; but

short-lived, so: Higher % of RNA synthesis devoted to mRNA


Prokaryotic mRNA One mRNA with a single promoter will

contain coding information for several proteins, i.e., 1 promoter, several genes

Defined stop codons show the ribosome where to put in the breaks

Translation closely coupled to transcription, unlike eukaryotic systems, where they’re separated in space & time


Eukaryotic mRNA One mRNA per protein But the mRNA will be initially synthesized with

noncoding segments (introns) interspersed between the coding segments (exons):heterogeneous nuclear RNA, hnRNA

snRNPs (q.v.) in nucleus splice out the introns, tying together the exons to make the mature transcript

Each mRNA will end with a poly(A) tail, added after transcription


Ribosomes and rRNA Ribosome is 65% RNA, rest protein Lots of intrastrand H-bonds Ribosomes characterized by

sedimentation coefficients E.coli: 50S piece+30S piece 70S total Eukaryotes 60S + 40S 80S total

rRNA has pseudouridine, ribothymidine, methylated bases


Prokaryotic ribosomes (fig.10.25a)


Eukaryotic ribosomes (fig. 10.25b)


Transfer RNA Each tRNA carries a specific

amino acid to the ribosomal protein synthesis machine

One full set of tRNA at each cellular site of protein synthesis (cytoplasm, mitochondrion, chloroplast)

These are small molecules: 55-94 bases



A/T sitetRNA model based on cryoEM complexPDB 1QZA


tRNA contents Many modified bases CCA on the 3’-end is

attached to the amino acid Catalytic attachment of

amino acid to protein is catalyzed by an adenine in one of the 50S rRNAs



Dieter Söll




Small nuclear RNAs snRNA found mostly

in nucleus 100-200 nucleotides closely associated with proteins

& with other RNA molecules Mostly in ribonucleoprotein particles

(snRNPs), which are involved in mRNA processing, converting full-length transcript into smaller transcript in which introns have been removed, leaving only the exons



Image courtesy Richard Lührmann,

Göttingen


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


iClicker question 3 Suppose you isolate an RNA molecule

that consists of 1500 bases. It is probably:

(a) tRNA (b) mRNA (c) rRNA (d) either mRNA or rRNA (e) none of the above.

Documents

10/02/08 Biochemistry: Nucleic Acid Chem&Struct Nucleic Acid Chemistry & Structure Andy Howard Introductory Biochemistry 2 October 2008