BCH3033 - CH 8 a.pdf

BCH3033: Biochemistry 1 Chapter 8 03.29.2024 Donella Beckwith, Ph.D. [email protected] 1 Nucleotides have a variety of roles in cellular metabolism: – energy currency in metabolic transactions – essential chemical links in the response of cells to hormones and other extracellular stimuli – structura...

BCH3033: Biochemistry 1 Chapter 8 03.29.2024 Donella Beckwith, Ph.D. [email protected] 1 Nucleotides have a variety of roles in cellular metabolism: – energy currency in metabolic transactions – essential chemical links in the response of cells to hormones and other extracellular stimuli – structural components of an array of enzyme cofactors and metabolic intermediates – constituents of nucleic acids: deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) 2 Nucleotides and Nucleic Acids Have Characteristic Bases and Pentoses nucleotides have three components: 1 – a nitrogenous base (pyrimidine or purine) 2 – a pentose 3 – 1 or more phosphates 1 3 2 nucleoside = the molecule without a phosphate group 3 Nucleotides and Nucleic Acids Have Characteristic Bases and Pentoses 1 – Nitrogenous base 3 1 4’ 3’ 2’ 1’ glycosidic bond 2 4 Question What other name can be given to a nucleotide? A. B. C. D. glycosylated nucleoside purinated pentose deoxyribonucleotide nucleoside phosphate 5 Nucleotide Bonds N-β-glycosyl bond = covalently joins the 1′ carbon of the pentose to the base (at N-1 of pyrimidines and N-9 of purines) – formed by removal of the elements of water What type of reaction is this? the phosphate is esterified to the 5′ carbon 6 Nucleotide Nitrogenous Bases major purine bases: – adenine (A) = in DNA and RNA – guanine (G) = in DNA and RNA major pyrimidine bases: – cytosine (C) = in DNA and RNA – thymine (T) = only in DNA – uracil (U) = only in RNA 7 Question Which of the following is NOT a pyrimidine base? A. B. C. D. cytosine uracil adenosine thymine 8 *Nucleotide and Nucleic Acid Nomenclature* Table 8-1 Nucleotide and Nucleic Acid Nomenclature Base Nucleoside Nucleotide Nucleic Acid Purines: Adenine Adenosine Deoxyadenosine Adenylate Deoxyadenylate RNA DNA Purines: Guanine Guanosine Guanylate Deoxyguanosine Deoxyguanylate RNA DNA Pyrimidines: Cytosine Cytidine Deoxycytidine Cytidylate Deoxycytidylate RNA DNA Pyrimidines: Thymine Thymidine or deoxythymidine Thymidylate or deoxythymidylate DNA Pyrimidines: Uracil Uridine Uridylate RNA 9 Nucleotide Pentoses two kinds of pentoses: – 2′-deoxy-D-ribose = in DNA – D-ribose = in RNA both are in their βfuranose (closed fivemembered ring) form 10 Question Which of the following is NOT part of a deoxyribonucleotide? A. B. C. D. a deoxyribonucleoside phosphate a nitrogenous base ribose 11 *Structure and Names of The Four Major Deoxyribonucleotides deoxyribonucleotides = structural units of DNA – also called deoxyribonucleoside 5′-monophosphates, deoxynucleotides, and deoxynucleoside triphosphates 2’ 12 *Structure and Names of The Four Major Ribonucleotides ribonucleotides = structural units of RNA – also called ribonucleoside 5′-monophosphates 2’ 13 Question What name is given to the nucleotide found in DNA and NOT in RNA? A. B. C. D. uridylate thymidine uracil deoxythymidylate 14 Minor Purine and Pyrimidine Bases Additional minor bases found in DNA (a) and RNA (b) methylated forms – most common in DNA 15 Nucleotides with Phosphate Groups in Different Positions ribonucleoside 2′,3′-cyclic monophosphates = isolatable intermediates ribonucleoside 3′-monophosphates = end products of RNA hydrolysis adenosine 3′,5′-cyclic monophosphate (cAMP) = regulates activity of protein kinase A guanosine 3′,5′-cyclic monophosphate (cGMP) = regulates activity of protein kinase G “Common Form” 16 Phosphodiester Bonds Link Successive Nucleotides in Nucleic Acids phosphodiester linkage = covalent bond that joins successive nucleotides of both DNA and RNA – between the 5′-phosphate group of one nucleotide unit and the 3′-hydroxyl group of the next nucleotide 17 Hydrolysis of DNA and RNA under alkaline conditions: – RNA is rapidly hydrolyzed due to the presence of 2′-hydroxyl groups – DNA is not rapidly hydrolyzed 18 Solubility of Nucleotides hydrophobic and relatively insoluble in pH 7.0 water – leads to stacking interactions (van der Waals and dipole-dipole) charged and more soluble at acidic or alkaline pH values 19 Question Why is DNA more stable than RNA? A. RNA can never be double-stranded; therefore, DNA is more stable. B. The 2′-hydroxyl groups in RNA (absent in DNA) are directly involved in the hydrolysis of oligonucleotides. C. Thymine forms more stable hydrogen bonds than uracil. D. RNA does not have secondary structure. 20 Schematic Representation of Nucleotide Sequences phosphate groups symbolized by ℗ deoxyribose symbolized by a vertical line, from C-1′ at the top to C-5′ at the bottom lines connecting nucleotides drawn diagonally from C-3′ to C-5′ 1’ 3’ 5’ 5’ 21 -Simpler Representations of Nucleotide Sequences- can also be written as: – pA-C-G-T-AOH – pApCpGpTpA – pACGTA p = penta = 5 22 Oligonucleotides and Polynucleotides oligonucleotide = short (typically < 50 nucleotides) nucleic acid segments – DNA or RNA – Single stranded – Utilized in genetic techniques such as Polymerase Chain Reaction (PCR) and DNA microarrays (done in a lab setting) polynucleotide = longer nucleic acid segments – DNA and RNA – Utilized in PCR and DNA sequencing 23 Question A strand of DNA or RNA: A. B. C. D. contains 5′ - 2′ phosphodiester bonds. might be an oligonucleotide. has a C-terminus and an N-terminus. has no charge. 24 The Properties of Nucleotide Bases Affect the Three-Dimensional Structure of Nucleic Acids weakly basic compounds aromatic molecules because most bonds in the ring have partial double-bond character: – pyrimidines are planar – purines have a slight pucker 25 Free Pyrimidine and Purine Bases May Exist as Tautomers may exist in readily interconverted forms called tautomers (structural isomers that interconvert) – lactam – lactim – double lactim predominates at pH 7.0 26 Absorption Spectra of Common Nucleotides all nucleotide bases absorb UV light Why? conjugated pi system of heterocyclic ring of base strong absorption near 260 nm 27 Question A property of nucleotide bases that affects the threedimensional structure of nucleic acids is: A. B. C. D. their weakly acidic nature. their absorption of UV light at wavelengths above 280 nm. their hydrophilic nature. their existence in tautomeric forms depending on pH. 28 Base Pairing Permits the Duplication of Genetic Information base pairs = hydrogen-bonding patterns between complementary strands of nucleic acids – A bonds specifically to T (or U) – G bonds specifically to C 29 Question In a sample of DNA isolated from an unidentified species of bacteria, adenine makes up 28% of the total bases. Which statement is false about the relative proportions of the bases in the DNA sample? A. B. C. D. The DNA must contain 28% C. A and T bases account for 56% of the bases. G and C base pairs account for 44% of the bases. C, T, and G bases account for 72% of the bases. 30 Hierarchical Levels of Nucleic Acid Structure primary structure = covalent structure and nucleotide sequence secondary structure = regular, stable structure taken up by some or all the nucleotides tertiary structure = complex folding of large chromosomes or the elaborate folding of tRNA or rRNA structures 31 Watson-Crick Model for the Structure of DNA DNA is a double helix that stores genetic information – X-ray diffraction pattern revealed DNA molecules are helical offset pairing of the two strands creates a major groove and a minor groove 3 hydrogen bonds form between G and C 2 hydrogen bonds form between A and T per turn 10.5 b.p. 32 DNA Strands Are Antiparallel parallel = 3′ ,5′ phosphodiester bonds run in the same direction antiparallel = 3′ ,5′ phosphodiester bonds run in opposite directions – ultimately confirmed by x-ray analysis 33 Question Which statement is true about the three-dimensional structure of DNA? A. Native DNA consists of two parallel chains in a righthanded double-helical arrangement. B. Complementary base pairs, A≡C and G=T, are formed by hydrogen bonding between chains in the helix. C. The base pairs are stacked parallel to the long axis of the double helix. D. The offset pairing of the two strands creates a major groove and a minor groove on the surface of the duplex. 34 Antiparallel Polynucleotides Chains Are Complementary double-helical DNA strands are complementary: – when A occurs in one chain, T is found in the other – when G occurs in one chain, C is found in the other hydrogen bonding does not contribute significantly to stability of the structure 35 Stabilization of the DNA Double Helix the double helix is stabilized by: – metal cations that shield the negative charges of backbone phosphates – base stacking interactions between successive base pairs successive G≡C or C≡G are stronger than successive A=T or T=A duplexes with higher G≡C context are more stable 36 Question Which statement regarding bases in DNA is false? A. B. C. D. They absorb UV light. Pyrimidines are aromatic; purines are not. They have vertical stacking interactions. They hydrogen bond between two or more complementary DNA strands. 37 Replication of DNA Step 1: preexisting or “parent” strands become separated Step 2: each “parent” strand serves as a template for the biosynthesis of a complementary “daughter strand” 38 DNA Can Occur in Different ThreeDimensional Forms structural variation in DNA reflects: – different possible conformations of the deoxyribose – rotation about the contiguous bonds making up the phosphodeoxyribose backbone – free rotation about the C-1′–N-glycosyl bond 39 The Three Forms of DNA B-form DNA = the Watson-Crick structure – most stable for a random-sequence DNA molecule under physiological conditions A-form DNA = right-handed double helix with a wider helix, 11 bp/turn, and a tilted plane – favored in solutions devoid of water Z-form DNA = left-handed helix with 12 bp/turn and a backbone with a zig-zag appearance – appears more slender and elongated 40 A, B, and Z Forms of DNA 41 Question Some effort has been made to extract DNA from fossilized dinosaur bones that have been buried for many millions of years in arid climates. In what form is this DNA MOST likely to be found? A. B. C. D. A B Z completely denatured 42 Certain DNA Sequences Adopt Unusual Structures palindrome = region of DNA that is identical when read either forward or backward – applied to regions of DNA with inverted repeats mirror repeat = sequence when the inverted repeat occurs within each individual strand (same side) 43 Hairpin and Cruciform Structures hairpin and cruciform structures = form from the selfcomplementarity within each strand 44 DNA Structures Containing Three DNA Strands Hoogsteen positions = N-7, O6, and N6 of purines – participate in the hydrogen bonding with a third DNA strand Hoogsteen pairing = the non-Watson-Crick pairing triplex DNAs = form from Hoogsteen pairing 45 DNA Structures Containing Four DNA Strands tetraplex DNAs = occur when four DNA strands pair – occur readily only for DNA sequences with a very high proportion of G residues – G tetraplex = very stable 46 Question Which of the following is NOT associated with DNA structure? A. B. C. D. Z-DNA G tetraplexes three hydrogen bonds between A and T pyrimidines in the anti conformation 47 Messenger RNAs Code for Polypeptide Chains “messenger RNA” (mRNA): portion of cellular RNA carrying the genetic information from DNA to the ribosome transcription = process by which mRNAs are formed on a DNA template 48 mRNA Can Be Monocistronic or Polycistronic monocistronic = codes for only one polypeptide – most mRNAs in eukaryotes polycistronic = codes for 2+ different polypeptides – occurs in bacteria and archaea noncoding RNA 49 Question Which statement regarding mRNA is false? A. B. C. D. It is always monocistronic. It carries genetic information to the ribosomes. It is formed by transcription. It can be of varying lengths. 50 Many RNAs Have More Complex Three-Dimensional Structures mRNA is always single-stranded – right-handed helical conformation – dominated by base-stacking interactions strongest between two purines – can base pair with complementary regions of DNA or RNA paired strands are antiparallel 51 Secondary Structure of RNAs structure of complementary RNA strands is an A-form righthanded double helix breaks caused by mismatched or unmatched bases result in bulges or internal loops internal loops form between palindromic sequences 52 Base-Paired Helical Structures In RNA extensive base-paired helical segments form in RNAs hairpins are the most common type of secondary structure Secondary structure 3D structure 53 Question Which structure is found in RNA but would be the result of a mutation of faulty annealing in DNA? A. B. C. D. hairpin double-stranded helix cruciform Bulge Annealing: when unwound segments of two strands spontaneously rewind = intact duplex 54 Double-Helical DNA and RNA Can Be Denatured denaturation, or melting, of the double helix: – due to pH extremes or high temperatures – disrupts hydrogen bonds and basestacking interactions 55 Question Which statement is false regarding a solution of DNA subjected to extremes of pH? A. B. C. D. The viscosity of the solution will decrease sharply. Hydrogen bonds between paired bases will be disrupted. Covalent bonds in the DNA will be broken. Base-stacking interactions will be disrupted. 56 Nucleotides and Nucleic Acids Undergo Nonenzymatic Transformations mutations = alterations in DNA structure that produce permanent changes in the genetic information encoded – linked to aging and carcinogenesis 57 Deamination Reactions deamination = spontaneous loss of exocyclic amino groups deamination of cytosine to uracil = ~100 events/day – recognized as foreign in DNA and removed – almost certainly why DNA contains thymine rather than uracil 58 Depurination Reactions depurination = hydrolysis of the N-β-glycosyl bond between the base and the pentose – creates an AP (apurinic, apyrimidinic) site or abasic site – more common with purines 59 Reactions Promoted by Radiation UV light causes: – cyclobutane pyrimidine dimers – 6-4 photoproduct ionizing radiation (x-rays and gamma rays) causes: – ring opening – base fragmentation – breaks in the covalent backbone of nucleic acids 60 Question Which statement is false about nucleotides and nucleic acids undergoing nonenzymatic transformations? A. Several nucleotide bases undergo spontaneous loss of their exocyclic amino groups. B. Hydrolysis of the N-β-glycosyl bond between the base and the pentose creates a DNA lesion called an AP site. C. Ionizing radiation induces the condensation of two ethylene groups to form a cyclobutene ring. D. UV irradiation can result in a 6-4 photoproduct, a type of pyrimidine dimer. 61 DNA Damage by Reactive Chemicals nitrous acid precursors = deaminating agents alkylating agents = generate modified nucleotides nonenzymatically 62 DNA Damage by Oxidative Damage reactive oxygen species (hydrogen peroxide, hydroxyl radicals, superoxide radical) damage DNA hydroxyl radicals are responsible for most oxidative DNA damage cells have an elaborate defense system to destroy reactive oxygen species 63 Question Mutations in DNA can result from: A. B. C. D. deamination of ribose. UV-induced purine dimers. alkylation of the phosphodiester bond. oxidation of deoxyribose. 64 Gene Sequences Can Be Amplified with the Polymerase Chain Reaction polymerase chain reaction (PCR): method of amplifying DNA segments of interest – relies on DNA polymerases (enzymes that synthesize DNA from deoxyribonucleotides (dNTPs) using a DNA template) – DNA polymerases add nucleotides to the 3′ ends of preexisting strands called primers 65 The Polymerase Chain Reaction 66 PCR Technology Is Highly Sensitive PCR can detect and amplify just one DNA molecule in almost any sample type What happens if contaminated (DNA specific answer)? Uses of PCR: – cloning of rare, undegraded DNA segments from 40,000+ years ago – tracing evolution – potent tool in forensic medicine – detecting viral infections and cancers before they cause symptoms – prenatal diagnosis of genetic diseases 67 Question In its simplest form, polymerase chain reaction: A. B. C. D. is remarkably insensitive to contamination. requires reverse transcriptase. requires two oligonucleotide primers. requires a minimum of 10,000 copies of DNA to be amplified. 68 The Sequences of Long DNA Strands Can Be Determined Sanger sequencing = dideoxy chaintermination sequencing nucleotide analogs called dideoxynucleoside triphosphates (ddNTPs) interrupt synthesis 69 Automation of DNA Sequencing Reactions each of the four ddNTPs is labeled with a differentcolored fluorescent tag 70 DNA Sequencing Technologies Are Advancing Rapidly “next-gen” sequencing: – reversible terminator sequencing – single-molecule real time (SMRT) sequencing 71 Reversible Terminator Sequencing developed by Illumina uses four different modified deoxynucleotides (A, T, G, and C), each with a particular fluorescent label and a 3′ blocking group 72 SMRT Sequencing SMRT sequencing = technology allowing read lengths averages up to 30,000-40,000 bp lower throughput, higher cost, and higher error rate than the Illumina approach 73 Sequence Assembly Requires the Computerized Alignment of Overlapping Fragments sequencing depth = the number of times that a particular nucleotide in a genome is sequenced, on average contigs = long, contiguous sequences that are assembled from overlaps 74 Nucleotides Carry Chemical Energy in Cells hydrolysis of nucleoside triphosphates provides chemical energy – ATP is the most widely used 75 Hydrolysis of Nucleoside Phosphates when coupled to a reaction with a positive free-energy change, ATP hydrolysis shifts the equilibrium to favor product formation – hydrolysis of the ester linkage yields ~14 kJ/mol (under standard conditions) – hydrolysis of each anhydride bond yields ~30 kJ/mol 76 Question Which statement is true? A. Hydrolysis of the α, β, and β, γ phosphoester bonds of a nucleotide is used as a source of chemical energy. B. dATP is the most common source of chemical energy in cells. C. CTP hydrolysis can be a source of chemical energy. D. Hydrolysis of ATP to ADP and phosphate releases about 14 kJ/mol of free energy. 77 Some Nucleotides Are Regulatory Molecules second messengers = compounds produced inside the cell following the interaction of extracellular chemical signals with receptors – often a nucleotide, like adenosine 3′,5′-cyclic monophosphate (cyclic AMP, or cAMP) ppGpp = produced in bacteria during amino acid starvation to inhibit the synthesis of the rRNA and tRNA molecules 78 Adenine Nucleotides Also Serve as Signals ATP and ADP serve as: – neurotransmitters in a variety of signaling pathways – signals for receptors that mediate pain sensation – blood clotting signals 79 Question In which way can nucleotides NOT be involved in signaling? A. B. C. D. cyclic dGMP as a second messenger as precursors for synthesizing second messengers extracellular ADP binding to its receptor extracellular ATP binding to its receptor 80