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Nucleosides, Nucleotides & Nucleic Acids

•  Genetic (heritable) information of the cell

•  Also have roles as – energy transfer molecules – cofactors/coenzymes

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Nucleotides •  Building block of the

nucleic acids •  3 characteristic

components – Nitrogen containing base – Pentose sugar – Phosphoryl group

•  If no phosphoryl group, then nucleoside

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Nitrogenous bases

•  Heterocyclic ring •  Two parent components

– Purine – Pyrimidine

•  Sugar link at N – N9 purines – N1 pyrimidines

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Pentose Sugar •  Pentose sugar in β-furanose

form •  Numbered by carbohydrate

convention –  Distinguished from base

numbering by ‘prime’ (´) •  Ring is puckered •  Linked to the nitrogenous base

at C1´ position –  N-β-glycosidic bond

•  Classification based upon which sugar is present –  Ribose or deoxyribose

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Phosphate Group •  Nucleotides have

anywhere from 1 to 3 phosphoryl groups

•  Linked to sugar •  Named by number of

phosophates –  Mono-, di-, tri-

•  Position of bonds identified by sugar position –  5’ –  2’3’ cyclic monophosphate

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Cellular Roles

•  Nucleic acid constituents – Genetic information – RNA, DNA

•  Components of enzyme cofactors •  Energy currency •  Act as messengers

– Link cellular responses to extracellular stimuli (I.e.hormones)

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Nucleic Acids •  Covalently linked nucleotides by

phosphoryl group bridges •  The 5’ phosphate linked to 3’ hydroxyl

of next nucleotide –  Phosphodiester linkages –  backbone is alternating phosphate and

pentose residues –  Nitrogenous bases, “side-group”, face out

at regular intervals •  Phosphate is completely ionized •  Sugar hydroxyl H-bonded to water •  Nitrogenous bases are hydrophobic

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Nomenclature

•  Oligonucleotide – Normally less

than 50 bases •  Polynucleotide

– Termed nucleic acid

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Directional molecule •  Phosphodiester links

have same orientation giving the linear nucleic acid specific polarity

•  Schematic representation via line drawing

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DNA vs RNA

•  Based upon the identity of the sugar •  Both subject to slow hydrolysis of the

phosphodiester bond •  Basic conditions will rapidly hydrolyze

RNA – Hydroxyl on 2’ position makes 3’

phosphodiester susceptible to hydrolysis

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Structure/Composition

•  DNA stores genetic information •  1868 first isolation of “nuclein”

– Fredrich Mieschner suspected that it was responsible for inheritance

•  1st direct evidence of DNA being heritable information molecule – Avery-MacLeod-McCarty experiment 1944 – Hershey-Chase experiment 1952

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Chargaff’s Rules •  Distinctive base composition noted •  Proposed in 1940’s

–  Base composition of DNA varies by species –  DNA from different tissues, but same species

same –  Base composition doesn’t change due to nutrition/

age/environment •  Regardless of species, for all cellular DNA

–  A=T; C=G –  A+G=C+T

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•  Non-direct evidence of DNA inheritance

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Hershey-Chase

•  Identification of which part of viral particle infects the cell

•  Direct evidence of material transferred

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Watson Crick Double Helix Structure

•  Knew prior information – Chargaff’s rules – Rosaline Frank/Maurice

Wilkins X-ray diffraction pattern •  Helices, two perodices along

long axis •  One 3.4 Å, 2nd 34 Å •  Proposed a model that co-

related all the information

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Model proposed in 1953 •  2 helical chains wound around same axis

– Right-handed double helix •  Hydrophilic backbone alternating

deoxyribose and phosphate groups on exterior – C2’ endo conformation of sugar

•  Hydrophobic and nearly planer nucleotide base close together – perpendicular to long axis

•  Pairing yields major and minor grooves

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•  Parallel or antiparallel strands? – Antiparallel yields two complementary

structures •  when A on one strand, T on opposite

– The complement •  Double helix held together by:

– hydrogen bonding between bases – base stacking interactions of the ring

structures •  Hydrophobic interactions of the planer rings

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3 forms of DNA •  DNA is a relatively flexible

molecule •  Flexibility due to

–  Possible conformations of the deoxyribose ring

–  Rotation about the contiguous bonds that make up the phosphodiester backbone

–  Free rotation about the C1´-N glycosyl bond

•  3 structural forms predominate –  A,B and Z

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B-form DNA •  Watson-Crick proposed

model •  Most stable under

physiological conditions •  Standard reference molecule •  Physical parameters

– 10.5 bases per turn – 3.4 Å rise per base – 34 Å per turn – Right-handed helix

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A-form DNA •  Favored in solutions devoid

of water •  Physical parameters

– 11 bases/turn – Base plane tilted

•  Deepened major groove •  Shallower minor groove

•  Uncertain if found physiologically

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Z-form DNA •  Radical departure from prior

structures •  Physical parameters

–  left-handed helix – 12 bp/turn – 2.7 Å rise per base – Elongated (zig-zag) appearance – Purine residues flip to the syn

conformation •  Found, role is uncertain

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Palindromic sequences •  Word or phrase that reads the same

forwards or backwards –  “Level, madam, level!"

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RNA structure •  Complex structures exist for RNA

molecules •  RNA molecule has more functions

within the cell – mRNA - messenger –  rRNA - ribosomal –  tRNA - transfer

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mRNA •  Messenger RNA carries genetic message

from the nucleus to the cytoplasm •  mRNA from different genes vary in length,

mRNA from a single gene has defined size – Monocistronic vs polycistronic

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•  Stem-loop structures – Both double and single

stranded areas

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rRNA •  Ribosomal RNA •  Jacob-Monod proposal

–  ribosomes were not manufactured anew each time a protein was made

–  the ribosomes did not contain the template necessary for the manufacture

–  Were structures that when supplied with the necessary building blocks and instruction (mRNA)

•  Conserved within organisms –  Most conserved gene –  16S/18S RNA allows taxonomic identification

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tRNA •  transfer RNA •  Adaptor molecule allowing interaction

between codon (nucleotide sequence) and amino acid

•  Has specific 3-dimensional structure –  Acceptor stem –  Anticodon stem-loop –  TΨC loop –  D (dihydrouridine) loop

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RNA and DNA can be denatured •  Denaturation - removal of H-

bonding –  Temperature, pH –  No covalent bonds broken

•  Renaturation called annealing •  One step

–  Rapid if molecules still associated at some point

–  “Zipping-up” of molecule •  Two step

–  Slower, molecules not associated –  First must associate, then have

regions of complementarity before the zip can occur

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Tm

•  Temperatures are content specific

•  G-C higher melting temp than A-T

•  Greater number of H-bonds

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Transformation •  Spontaneous loss of groups

–  Deamination •  exocyclic amino group

–  Depurination •  Loss of nitrogenous bases •  Hydrolysis of N-b-glycosyl bond higher for purines than

pyrimidines

•  Pyrimidine dimers induced by UV irradiation –  Formation of a cyclobutyl ring between two adjacent thymines

•  Methylation –  Addition of a methyl (-CH3) group –  Adenine and cytodine more often methylated –  S-adenosylmethionine methyl group donor

•  Mutation –  If in DNA, daughter chains will have permanent change to

the sequence

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Sanger dideoxy sequencing •  Requires

– Template – Primer – All four dNTPs +

ddNTPs radiolabeled – Enzyme (polymerase)

•  Label incorporated is complementary to base present on template

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Energy Currency •  Phosphoanhydride bond

between phosphoryl groups in nucleotidetriphosphates is a high energy bond –  Have α, β, γ phosphoryl groups –  The α-β, β-γ are

phosphoanhydride –  Sugar to α is a ester

•  Most common carrier of energy is the adenine base

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Enzyme cofactors •  Have nucleotide as

part of the structure •  Nucleotide doesn’t

participate in the function, but acts as a handle, allowing binding energy to participate in enzyme-substrate interaction

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Regulatory molecules/messengers •  Serve to modulate the

activity of enzymes/pathways

•  Second messengers as they are produced in the cell in response to message from outside of the cell

•  Commonly cyclic AMP (cAMP, 3’5’cAMP) and ppGpp