Lecture 3 Nucleic Acids and DNA PDF

Lecture 3 Nucleic acids and DNA BIO206 Introductory Cell & Molecular Biology Instructor: Ichiro Inamoto University of Toronto Mississauga 1 Table of contents 3.1 Nucleic Acids 3 3.2 DNA is a polymer of nu...

Lecture 3 Nucleic acids and DNA BIO206 Introductory Cell & Molecular Biology Instructor: Ichiro Inamoto University of Toronto Mississauga 1 Table of contents 3.1 Nucleic Acids 3 3.2 DNA is a polymer of nucleotides 19 3.3 Properties of the DNA double helix 40 2  Lecture 3.1 Nucleic Acids 3  Central Dogma of Molecular Biology Storage and inheritance of genetic information Processing genetic information to build proteins Proteins built according to genetic Cellular activity performed by proteins: information. Catalyze metabolic reactions Build cellular structures Send / receive signals and much more 4 Nucleotide Functions Building blocks of DNA – Storage of genetic information Building blocks of RNA – carrier of genetic information + structural components of tRNA, ribosomes, spliceosomes etc Energy metabolism – ATP, GTP perform biochemical work Cellular signalling – second messengers in the form of cAMP etc. 5 Nucleotide Structure Deoxyribonucleotides DNA Ribonucleotides RNA DNA is a linear polymer of deoxyribonucleotides RNA is a linear polymer of ribonucleotides 6 Nucleotides are composed of: phosphate + pentose sugar (5 C) + nitrogenous base 2 types of 5-carbon sugars either ribose or deoxyribose 5 different nitrogen-containing bases Adenine (A) Guanine (G) Thymine (T) or Uracil (U) Cytosine (C) phosphate group maximum of three nucleotide 7 Nucleotides are composed of: phosphate + pentose sugar (5 C) + nitrogenous base 2 types of 5-carbon sugars either ribose or deoxyribose 5 different nitrogen-containing bases Adenine (A) Guanine (G) 1 – 3 phosphates Thymine (T) or Uracil (U) (triphosphate) Cytosine (C) nitrogenous base phosphate group pentose sugar (adenine) maximum of three (ribose) nucleotide 8 Nucleotides vs. nucleosides phosphates + sugar + base = nucleotide sugar + base = nucleoside nucleotide nucleoside nucleotide 9 Nucleotide variation 1: Contains up to three phosphates monophosphate diphosphate triphosphate no phosphate group Nucleotide Nucleoside 10  Nucleotide variation 2: Five types of nitrogenous bases Nitrogenous bases either have a single ring or a double ring structure Pyrimidines have single ring Purines have a double ring Longer name, smaller structure Shorter name, larger structure 11 Nucleotide variation 2: Five types of nitrogenous bases Pyrimidines have six atoms constituting its ring, numbered 1 – 6 Two nitrogens at positions 1 and 3 Carbon 2 always connected to an oxygen with a double bond Carbon 4 is always connected to an extra atom outside the ring Above is true at least for the three common Three types of pyrimidines pyrimidines used in the central dogma. Other types Thymine (T), Cytosine (C), Uracil (U) of pyrimidines may not follow the same rule. 12 Nucleotide variation 2: Five types of nitrogenous bases Purines have nine atoms constituting its two rings, numbered 1–9 Nitrogen in the larger ring furthest away from the smaller ring is nitrogen 1 Nitrogen in the smaller ring connected to carbon 5 is nitrogen 7 Nitrogen in the smaller ring without a double bond is nitrogen 9 Two types of purines Above is true at least for the two common purines used in the central dogma. Other types of purines Adenine (A), Guanine (G) 13 may not follow the same rule. Nucleotide variation 3: Two types of Pentose Sugars H Has OH group at the #2 carbon Lacks OH group at the #2 carbon Building block of Ribonucleic Acid Building block of Deoxyribonucleic Acid 14  Numbering atoms in nitrogenous bases and sugars Carbons and nitrogens of nitrogenous bases and pentose sugars are numbered 7 4 Purines are numbered 1 – 9 6 5 3 5 1 Pyrimidines are numbered 1 – 6 8 2 6 2 4 Ribose sugars are numbered 1' – 5' 3 9 1 Numbers of the ribose sugar are noted with a prime to distinguish from the numbers of the nitrogenous bases 15  Numbering atoms in nitrogenous bases and sugars The sugar and the base are linked by an N-glycosidic bond 7 6 4 5 3 5 1 8 Bond-forming atoms are consistent 2 4 2 6 3 9 1 Purines C1’ – N9 N-glycosidic bond C1’ – N1 N-glycosidic bond for purines for pyrimidines Nitrogen 9 is connected to the 1' Carbon of ribose sugar Pyrimidines Nitrogen 1 is connected to the 1' Carbon of ribose sugar 16 Nucleoside triphosphate structure: Adenosine triphosphate phosphate attached on 5’ carbon Adenine    (Base) 9 5’ N-Glycosidic 1’ 4’ Ribose (Sugar) 2’ 3’ Adenosine (nucleoside) AMP ADP ATP 17 Nomenclature Note the difference between AMP & dAMP or CTP & dCTP etc. (not shown) Name of a nucleotide = ‘name of nucleoside’ + number of phosphates 18  Lecture 3.2 DNA is a polymer of nucleotides 19 DNA / RNA – linear polymer of nucleotides Deciphering DNA structure 20 Nucleotides polymerize! 21 Nucleotides attach to each other through 5’ and 3’ carbons phosphate attached to 5’ carbon Adenine    (Base) 9 5’ N-Glycosidic C2’- deoxyribose 1’ 4’ Ribose (Sugar) 2’ C2’ & C3’ - 3’ dideoxyribose Adenosine (nucleoside) AMP new bases ADPattach to the 3’ carbon via single phosphate group 22 ATP  DNA / RNA – linear polymer of nucleotides Polymerization occurs via formation of 3’ to 5’ phosphodiester bonds 3' carbon of the 'first' nucleotide attacks the 5' carbon of the 'second' nucleotide This is an enzyme catalyzed condensation reaction 5’ hydrolysis + 3’ H 2O 3’ carbon 5’ 5’ carbon NTP 3’ 23 DNA gets synthesized in the 5’ → 3’ direction Nitrogenous Base Base Base nucleotide 1 O 5' 4' P P P Sugar P P P C C C 1' Sugar 3' OH Base nucleotide 2 3' C C 2' 3’ to 5’ phosphodiester 5' bond formation P P P Sugar O H OH nucleotide 1 is about to polymerize with the nucleotide 2 3' OH of the nucleotide 1 attacks the 5' phosphates of nucleotide 2 24  DNA gets synthesized in the 5’ → 3’ direction Nitrogenous Base 1 Base Base O 2 5' 4' P P P Base Sugar P P P C C C 1' Sugar P Sugar 3' C C 2' OH P P + H2O O H nucleotide 1 is about to polymerize pyrophosphate and water gets released as the result of polymerization with the nucleotide 2 3' OH of the nucleotide 1 attacks the 5' phosphates of nucleotide 2 3' – 5' phosphodiester bond connects nucleotide 1 and 2 25 DNA gets synthesized in the 5’ → 3’ direction Nitrogenous Base 1 Base Base O 2 5' 4' P P P Base Sugar P P P C C C 1' Sugar P Sugar 3' C C 2' 3' OH Base nucleotide 3 O 5' H P P P Sugar OH nucleotide 3 is about to polymerize 3' OH of the nucleotide 2 attacks the 5' phosphates of nucleotide 3 26 DNA gets synthesized in the 5’ → 3’ direction Nitrogenous Base 1 Base Base O 2 5' 4' P P P Base Sugar P P P C C C 1' 3 Sugar P Sugar Base 3' C C 2' P Sugar O 3' OH Base nucleotide 4 H 5' P P P Sugar OH Incoming nucleotide ALWAYS gets added to the 3' end of the already existing poly-nucleotide 27 DNA gets synthesized in the 5’ → 3’ direction Nitrogenous Base 1 Base Base O 5' 2 5' 4' P P P Base Sugar P P P C C C 1' 3 Sugar 5’ P Sugar Base 4 3' C C 2' P Sugar Base 5 O P Sugar Base H 6 P Sugar Base The polynucleotide starts at the 5' 7 carbon of nucleotide 1 P Sugar Base 8 The polynucleotide has an open 3' OH P Sugar Base on the opposite side which is ready to accept more nucleotides P Sugar 3' DNA always grows in the 5' to 3' 3’ OH direction 28 DNA gets synthesized in the 5’ → 3’ direction Nitrogenous Base Base Base O 5' 5' 4' P P P Base Sugar P P P C C C 1' Sugar 5’ P Sugar Base 3' C C 2' P Sugar Base O P Sugar Base H P Sugar Base Notice the alternating chains of sugar and phosphate. P Sugar Base This is called the sugar-phosphate P Sugar Base backbone. P Sugar Also, the 5' end of the linear 3' polynucleotide will always have three 3’ OH phosphates attached 29 Polynucleotide has a (nearly) uniform, negative charge 1 One inorganic phosphate connects one Base nucleoside to the 3' end of another - - - 5' 2 nucleoside - P P P Sugar Base 3 Inorganic phosphates in the sugar 5’ - P Sugar Base 4 phosphate backbone has one negative charge - P Sugar Base 5 Therefore, a polynucleotide always has a - P Sugar Base 6 nearly uniform, negative charge where the number of bases is nearly equal to the - P Sugar Base 7 number of negative charges the only exception is the 5' end of the - P Sugar Base 8 molecule, where there may be more than one negative charge per base - P Sugar Base however in many cases, this exception is negligible during analysis since - P 3' Sugar many nucleotides are over 100s of bases long 3’ OH 30 DNA is (usually) double stranded 31  Watson Crick base pairing Complementary base pairing nitrogenous bases forms a pair via hydrogen bonds Adenine pairs with a Thymine (or Uracil) Has two hydrogen bonds in between Guanine pairs with a Cytosine Has three hydrogen bonds in between Purine pairs with a pyrimidine 32  Nucleotides polymerize and form double stranded DNA Base-pairing of nitrogenous bases between two strands of DNA holds the strands together (Double stranded DNA) Two strands are held antiparallel to each other with respect to their 5' – 3' polarity Sugar-phosphate backbone on the outside Nitrogenous bases inside, forming Watson Crick base-pairs 33  Nucleotides polymerize and form double stranded DNA Base-pairing of nitrogenous bases between two strands of DNA holds the strands together (Double stranded DNA) Two strands are held antiparallel to each other with respect to their 5' – 3' polarity Sugar-phosphate backbone on the outside Nitrogenous bases inside, forming Watson Crick base-pairs 34  Base pairing is always purine + pyrimidine Distance between the two C1’ carbons in a fits nicely base-pair = 10.85 Å (1.085 nm) not enough space Only purine-pyrimidine pairs fit in this distance without distorting the too far for H- bond formation double helix backbone while maintaining H- bond distances ~ 10.85 Å Central axis 35  Hydrogen bonds hold DNA strands together two H-bonds C – G pair has one more hydrogen bond compared to A– T pair Takes more energy to break apart a CG pair Denaturation hydrogen bonds break with heat How much GC pair a genome has as opposed to AT (% GC of genome) is variable for different organisms Species living in extremely high temperatures may have higher % GC 36 three H-bonds  Hydrogen bonds hold DNA strands together Chemical implication: For every Adenine, you have a Thymine For every Guanine, you have a Cytosine %A = %T, and %G = %C Q: An organism’s genome is 15 % Adenine. What is the % Guanine for this organism? 1) ‘15 % A’ = this organism has ‘15 % T’ 2) Total amount of A + T is 30 % 3) The rest of the genome must be G + C = 100 % – 30 % = 70 % 4) Half of 70 % is C = 70/2 = 35 % of the genome is C 37  Implications of base pairing Base-pairing rule has huge impact on how genetic material is passed on to the next generation It’s easy to ‘copy’ DNA using a single strand of parental DNA as a TEMPLATE Cells use this property to replicate DNA Scientists use this to make copies of DNA in PCR, etc. 38 Writing a DNA / RNA sequence 5’ 3’ 3’ 5’ This DNA sequence will be written as: CATTGCCAGT alternatively: ACTGGCAATG Convention is to write nucleotide sequence from 5’ → 3’ Base pair abbreviations A, T (U), C, G Show one strand only Only seeing sequence of one strand does not mean that the complementary strand does not exist 39  Lecture 3.3 Properties of the DNA double helix 40 single-stranded DNA = ssDNA double-stranded DNA = dsDNA dsDNA is helical 41 DNA / RNA – linear polymer of nucleotides Deciphering DNA structure 42  Rosalyn Franklin’s x-ray diffraction image of DNA Franklin and Wilkins analyzed DNA by bombarding DNA crystals with X-rays. Their analysis yielded two numbers that sparked interest—3.4 nm and 0.34 nm 43  2 nm Rosalyn Franklin’s x-ray diffraction image of DNA DNA is twisted helically 3.4 nm = Distance per one turn of double helix 3.4 nm 0.34 nm = Distance between adjacent base-pairs, therefore: 0.34 nm 3.4 nm in one turn = 10 bases / turn 0.34 nm between bases Diameter of the helix = 2 nm Distance between two C1’ carbons in base pair = 1.085 nm 44 Forces holding DNA strands together 0.34 nm 1.085 nm 2 nm 45 DNA is a right-handed helix 46  Three structural variants of dsDNA: A, B & Z A-DNA: right handed short and broad