Nucleotides And Nucleic Acids: Lehninger

Nucleotides and Nucleic Acids

Lehninger Chapter 8

BCH261 W 2015

1

CHAPTER 8 Nucleotides and Nucleic Acids Key topics: Biological function of nucleotides and nucleic acids Structures of common nucleotides Structure of double stranded DNA Structures of ribonucleic acids Denaturation and annealing of DNA Chemistry of BCH261 nucleic W 2015acids; mutagenesis 2

So far….. Monomer Amino Acids Monosaccharides

Nucleotides

Oligomer Peptides

Polymer Polypeptides (proteins)

Oligosaccharides

Polysaccharides (glycans)

Oligonucleotides

Polynucleotides (nucleic acids) Macromolecules in cells

BCH261 W 2015

3

Functions of nucleotides and nucleic Acids Energy for metabolism (ATP)

Nucleotide Functions

Enzyme cofactors (NAD+) Signal transduction (cAMP) Storage of genetic info (DNA)

Nucleic Acid Functions

Transmission of genetic info (mRNA) Protein synthesis (tRNA and rRNA) Processing of genetic information (ribozymes)

BCH261 W 2015

4

Two major functions of nucleotides and nucleic Acids nucleic acids

nucleotide  Adenosine triphosphate (ATP)

BCH261 W 2015

 Deoxyribonucleic acid (DNA)  Ribonucleic acid (RNA)

5

The anatomy of a nucleotide Phosphate

BCH261 W 2015

6

Nucleobases  Derivatives of pyrimidine or purine  Pyrimidine is single-ringed  Purine is double-ringed  Nitrogen-containing heteroaromatic molecules  Planar ring structures  Absorb UV light around 250-270 nm fig 8­1b

BCH261 W 2015

7

Pyrimidine bases

(DNA and RNA)

(DNA only)

(RNA only)

• All are good H-bond donors and acceptors • Neutral at neutral pH BCH261 W 2015

8

Purine bases

BCH261 W 2015

 Both found in DNA and RNA  Also good H-bond donors and acceptors 9

 Neutral at neutral pH

UV absorption of nucleobases  Absorption of UV light at 250-270 nm  This is due to ____________________  Excited states of common nucleobases decay rapidly via radiation-less transitions  Effective photoprotection of genetic material  No fluorescence from nucleic acids 

Used to measure nucleic acid concentration

Fig. 8-10, Lab 8

BCH261 W 2015

10

Absorbance of nucleotides is slightly different that that of aromatic amino acids Aromatic amino acids

absorbance

Nucleotides

240

250

260

270

Wavelength (nm)

BCH261 W 2015

280

290 11

Pentose in nucleotides  -D-ribofuranose in RNA  -2’-deoxy-D-ribofuranose in DNA

BCH261 W 2015

12

Conformation of Ribose

BCH261 W 2015

13

Ribose rings are not planar: different puckered conformations of the sugar ring are possible

C-2’ or C-3’ are the carbons that undergo bendin BCH261 W 2015

14

Joining the base to the pentose The N-Glycosidic Bond

BCH261 W 2015

15

 N-Glycosidic bond  The pentose ring is attached to the nucleobase via a N-glycosidic bond  The anomeric carbon of the sugar is in  configuration  It forms a bond with position N1 in pyrimides and position N9 in purines

9

1

 Free rotation in free nucleotides

• Stable, but hydrolyzable by acid BCH261 W 2015

16

Joining the phosphate to the pentose The phosphoester bond

BCH261 W 2015

17

Phosphate group

No phosphate : One phosphate: Two phosphates: Three phosphates: BCH261 W 2015

18

Nucleoside triphosphates

BCH261 W 2015

19

Nucleotides and phosphate number

NMP = NUCLEOSIDE MONOPHOSPHATE NDP = NUCLEOSIDE DIPHOSPHATE NTP = NUCLEOSIDE TRIPHOSPHATE

BCH261 W 2015

20

Nomenclature

BCH261 W 2015

21

Nomenclature: Ribonucleotides Recognize general structure, and the specific names and symbols (both one-letter (A) and three-letter (AMP) codes)

Nomenclature: Deoxyribonucleotides Recognize general structure, and the specific names and symbols (both one-letter (dA) and three-letter (dAMP) codes)

Joining nucleotides together: the phosphodiester bond

BCH261 W 2015

24

Nucleic Acids  Nucleic acids are made by covalently bridging nucleosides with a phosphodiester bond  Phosphate links the 3’ end of one nucleoside to the 5’ end of another  Phosphodiester bonds are not fully extended with respect to bases and sugars

BCH261 W 2015

25

Nucleic Acid Synthesis  The monomers used for nucleic acids are (d)NTPs!  Formation of Phosphodiester bond is directional!  A nucleotide is added to the 3’ end of an existing chain  Formation of phosphodiester bonds results in release of pyrophosphate  No branching  Polynucleotide chain is directional: exposed 5’ and 3’ ends  Always read and write sequence from 5’ to 3’ end BCH261 W 2015

26

Two types of nucleic acids

Fig. 8­7

BCH261 W 2015

27

Some properties of RNA and DNA  Covalent bonds formed via phosphodiester linkages  negatively charged backbone

 Linear polymers  No branching or cross-links

 Directionality  5’ end is different from 3’ end  We read the sequence from 5’ to 3’

 DNA backbone is fairly stable  DNA from mammoths?  Hydrolysis accelerated by enzymes (DNAse)

 RNA backbone is unstable  In water, RNA lasts for a few years  In cells, mRNA is degraded in few hours  Degraded by Alkali conditions

 DNA is a helix and RNA is typically not BCH261 W 2015

28

Watson-Crick model of B-DNA

BCH261 W 2015

29

rall DNA structure with GC and AT base p

BCH261 W 2015

30

What drives DNA HELIX formation? 1.Phosphate and pentose are hydrophillic - exposed to water - forms a hydration shell

2. Hydrogen bonding between bases - between bases on opposite strands

3. Base stacking - bases are hydrophobic rings - bases are planar - adjacent bases in the same strand are able to stack very closely BCH261 W 2015

31

Hydrogen bonding interactions (1)  Two bases can hydrogen bond to form a base pair  In double-stranded DNA, Watson-Crick base pairs predominate  A pairs with T  C pairs with G

 Purine pairs with pyrimidine

BCH261 W 2015

32

Stick model of a GC base pair showing H-bond

BCH261 W 2015

33

Stick model of a AT base pair showing H-bonds

BCH261 W 2015

34

Base stacking interactions

BCH261 W 2015

35

Chemistry reminder: stacking interaction between aromatic rings

 Pi stacking (also called π–π stacking) refers to attractive, noncovalent interactions between aromatic rings.  These interactions are important in nucleobase stacking within DNA and RNA molecules, protein folding, and molecular recognition BCH261 W 2015

36

DNA can be visualized with fluorescent dyes: this takes advantage of the ring stacking interactions in DNA. Base 1’

Base 1

Base 2’

Base 2

Ethidium bromide Excited by UV, and fluorescent only when bound to DNA BCH261 W 2015

37

Complementarity of DNA strands  Two chains run ________  Two chains are __________ together  Two chains are _______ in sequence (sequence is read from 5’ to 3’)

BCH261 W 2015

38

Discovery of DNA structure 

You have or will learn about the discovery of the DNA helix in other courses: Genetics, Molecular Biology



Elucidated by Watson and Crick



They used X-ray data obtained by Franklin and Wilkins



One of the most important discoveries in biology. Why?

BCH261 W 2015

39

Replication of Genetic Code  Strand separation occurs first  Each strand serves as a template for the synthesis of a new strand  Synthesis is catalyzed by enzymes known as DNA polymerases  Newly made DNA molecule has one daughter strand and one parent strand. BCH261 W 2015

40

NA can assume different conformatio

The B-form of DNA is the most common form, However Z-DNA is also very important. Z-DNA is found in regions of DNA that are actively being transcribed, and we have proteins that specifically recognize Z-DNA in order to facilitate transcription.

BCH261 W 2015

41

What causes DNA to assume different conformations? 1. Rotation about the Phosphate and Phosphodiester bonds in the backbone 2. Rotation of the C-1’-Nglycosyl bond 3. Conformation of deoxyribose (pucker of the 2’ or 3’ position)

BCH261 W 2015

42

cal characteristics of different DNA f

BCH261 W 2015

43

Describing DNA sequences Always write sequence in 5’ - > 3’ direction: Left strand:

Right strand:

BCH261 W 2015

44

Unusual DNA sequences Stretches of six adenosines can bend DNA 18°  Palindrome sequences Palindromes: words or phrases that are the same when read backward or forward: ROTATOR

BCH261 W 2015

NURSES RUN

45

Self-complementary within same strand

BCH261 W 2015

46

Palindrome DNA can form hairpins and cruciform conformations

May regulate gene regulation or BCH261 W 2015 demarcate specific

47

DNA Denaturation  Covalent bonds remain intact  Genetic code remains intact  Hydrogen bonds are broken  Two strands separate  Base stacking is lost  Denaturation can be induced by high temperature, or change in pH  Tm = melting temperature at which half of DNA molecules are denatured  Denaturation may be reversible: __________ BCH261 W 2015

48

Body temperature

Body temperature

Decrease temperatu re further

Increase temperat ure

Increase temperatur e further

Decrease temperatu re

BCH261 W 2015

49

DNAs with different sequence composition differ in melting temperature

DNA 1 DNA 2

BCH261 W 2015

50

Factors Affecting DNA Denaturation • The midpoint of melting (Tm) depends on base composition – high CG increases Tm

• Tm depends on DNA length – Longer DNA has higher Tm – Important for short DNA

• Tm depends on pH and ionic strength – High salt increases Tm BCH261 W 2015

51

Higher GC content increases Tm of DNA helix

BCH261 W 2015

52

• Denaturation of VERY Large DNA Molecules is not Uniform • AT rich regions melt at a lower temperature than GC-rich regions

Helix

Melted Helix

BCH261 W 2015

53

Molecular Mechanisms of DNA Mutagenesis

BCH261 W 2015

54

Molecular mechanisms of mutagenesis  Spontaneous mutagenesis  Deamination (enhanced by certain chemicals)  Depurination (enhanced by certain chemicals)

 Chemical alkylation  Methylation of guanine

 Oxidative damage  Hydroxylation of guanine  Mitochondrial DNA is most susceptible

 Radiation damage BCH261 W 2015

55

Deamination

Deamination • Very slow reactions • Large number of residues • The net effect is significant: 100 C  U events /day in a mammalian cell

BCH261 W 2015

56

Depurination

Depurination  N-glycosidic bond is hydrolyzed  Significant for purines:  10,000 purines lost/day in a mammalian cell

BCH261 W 2015

57

Radiation-Induced Mutagenesis • UV light induces dimerization of pyrimidines, this may be the main mechanism for skin cancers • Ionizing radiation (X-rays and -rays) causes ring opening and strand breaking. • Cells can repair some of these modifications, but others cause mutations. Accumulation of mutations is linked to aging and carcinogenesis BCH261 W 2015

58

RNA  Is synthesized using DNA template  Contains ribose instead of deoxyribose  Contains uracil instead of thymine  Can contain modified nucleosides  Can form DNA/RNA hybrids  Can form RNA double helices (viruses)  Can form complex tertiary structures  Several types of RNA  Can have unusual base pairs (eg. G-U) BCH261 W 2015

59

Conformations that RNA can adopt

Messenger RNA: code carrier for the sequence of proteins  Is synthesized using DNA template:  TRANSCRIPTION  mRNAs encode for proteins  TRANSLATION  Stabilized by base stacking instead of H-bonding but can form base pairs BCH261 W 2015

61

Transfer RNA: matching amino acids with the mRNA Code tRNA molecules have quite complex structures Where the amino acid goes

Where the anti-codon is – interacts with mRNA codon BCH261 W 2015

62

RNA and mRNA interaction is driven by base pa

tRNA anticodon

5’ A A G 3’ 3’ U U U 5’ BCH261 W 2015

Codon - mRNA

63

Other structural and enzymatic RNAs • Ribosomal RNAs (rRNAs) molecules have quite complex structures. Part of the protein synthesis machinery • Micro RNAs (miRNAs) molecules are involved in gene regulation • Small nuclear RNAs (snRNAs) molecules are involved in catalysis, intron splicing • Enzymatic RNAs are called ribozymes BCH261 W 2015

64

• Ribosomal RNAs (rRNAs) molecules have quite complex structures. Part of the protein synthesis machinery

http://upload.wikimedia.org/wikipedia/commons/e/e4/50SBCH261 W 2015 65 subunit_of_the_ribosome_3CC2.png

Nucleotide functions 1. Energy carriers

2. Co-enzymes

3. Signaling molecules

BCH261 W 2015

66

BCH261 W 2015

ATP is the main energy carrier in cells used to drive endergonic reactions

8­37

 Nucleotides are used as energy carriers in cells: ATP is predominant, but GTP, UTP and CTP are also used in some reactions  Phosphoanhydride bonds are high energy: 30 kJ/mol/anhydride 67  Coupled to synthetic reactions to drive endergonic

Nucleotides can function as co-enzymes NAD+ used by many enzymes in redox reactions (Vitamin B3/ Niacin)

e.g. Alcohol dehydrogenase

BCH261 W 2015

68

Nucleotides can function as co-enzymes

(Vitamin B5)

It is a coenzyme, notable for its role in the synthesis and oxidation of fatty acids, and the oxidation of pyruvate in the citric acid cycle

Nucleotides can function as coenzymes (Vitamin B2)

 FAD is a redox cofactor, more specifically a prosthetic group, involved in several important reactions in metabolism.  Very important in the process known as oxidative phosphorylation (a topic for BCH361).

Nucleotides can function as signaling molecules hormone

cAMP is derived from ATP It is a second messenger – signaling molecule

ATP

Adenyla te cyclase

cAMP Fig. 8-39

Hormon e actions BCH261 W 2015

71

Chapter 8: Summary  Function of nucleotides and nucleic acids  Names and structures of common nucleotides  Structural basis of DNA function  Reversible denaturation of nucleic acids  RNA structures and functions  Aspects of mutagenesisQuestions, 6e Questions in blue would be good exam questions. BCH261 W 2015

1, 2, 3, 5, 8 10, 12 Questions 5e 1, 2, 3, 5, 7, 8,10 72