Nucleic Acids and Nucleotides_2024.pdf

BCCB2000 Foundations of Biochemistry Nucleic acids and nucleotides Ricardo L. Mancera WARNING This material has been reproduced and communicated to you by or on behalf of Curtin University in accordance with section 113P of the Copyright Act 1968 (the Act) The material in this communication may be s...

BCCB2000 Foundations of Biochemistry Nucleic acids and nucleotides Ricardo L. Mancera WARNING This material has been reproduced and communicated to you by or on behalf of Curtin University in accordance with section 113P of the Copyright Act 1968 (the Act) The material in this communication may be subject to copyright under the Act. Any further reproduction or communication of this material by you may be the subject of copyright protection under the Act. Do not remove this notice. 1 Learning objectives  When you complete this lecture you will be able to  Understand the importance of nucleotides in structure and function  Describe the structure of nucleotides  Describe the structure and properties of DNA  Describe the structure and properties of RNA  Understand the functional and structural relationship between DNA and RNA  Use the biochemical knowledge learned to answer questions and solve problems Importance of nucleotides  Precursors of nucleic acids   Precursors of ‘energy carriers’   e.g. NAD, FAD, S-adenosylmethionine, coenzyme A Components of activated intermediates   ATP and GTP Components of cofactors   DNA and RNA e.g. UDP-glucose and CDP-diacylglycerol Metabolic regulators (second messengers)  e.g. cyclic AMP (cAMP) and cyclic GMP (cGMP) 2 Nucleotides Nitrogenous bases All of these bases are planar molecules Pyrimidines O H C 4 N3 HC They are weak bases because the nitrogen can act as a base (proton acceptor) due to its lone electron pair C CH HN CH 1 N C C O Parent Structure CH3 O NH2 C C HN CH CH C O N H Thymine N CH CH C O N H CH N H Cytosine Uracil Purines N1 HC H C 6 3 N O NH2 C C N 7 9 C CH N H Parent Structure N C C N CH HC C N Adenine N H C HN N CH H 2N C C N N H Guanine 3 Sugars in nucleosides Ribose and Deoxyribose are pentose sugars Ribose in RNA Deoxyribose in DNA No oxygen Note: The atoms in the sugar of a nucleoside or nucleotide are labelled with an apostrophe, e.g. 5’ and pronounced ‘5 prime’. This is to distinguish them from the atoms in the base. Ferrier 2014 Phosphates in nucleotides Bases in DNA Adenine Guanine Cytosine Thymine Bases in RNA Adenine Guanine Cytosine Uracil Ferrier 2014 4 Phosphates in nucleotides NH2 O Carbon ester bond R1 C O R2 N O Phosphate ester bond R1 P Ester O R2 O Ester N O O N Ester N O Anhydride P -O O O- P O O- P O O OH phospho-anhydrides ‘R1’ and ‘R2’ are the rest of the molecule that is not a hydrogen H H OH H OH glycosidic bond Adenosine triphosphate (ATP) Naming of bases, nucleosides and nucleotides Base Ribonucleoside Base + Ribose Ribonucleotide Base + Ribose + phosphate (5’ ribonucleotide) Adenine (A) Adenosine Adenylate (AMP) Guanine (G) Guanosine Guanylate (GMP) Uracil (U) Uridine Uridylate (UMP) Cytosine (C) Cytidine Cytidylate (CMP) Base Deoxyribonucleoside Base + deoxyribose Deoxyribonucleotide Base + deoxyribose + phosphate (5’ deoxyribonucleotide) Adenine (A) Deoxyadenosine Deoxyadenylate (dAMP) Guanine (G) Deoxyguanosine Deoxyguanylate (dGMP) Thymine (T) Dexoythymidine Deoxythymidylate (dTMP) Cytosine (C) Deoxycytidine Deoxycytidylate (dCMP) 5 Nucleic acids Nucleic Acids Oligonucleotide (20 nucleotides) DNA Guanine Purine Base Pyrimidine Base Adenine Thymine deoxyRibose Cytosine RNA Phosphate Purine Base Pyrimidine Base Ribose Guanine Adenine Uracil Cytosine Phosphate Nucleic acids – Deoxyribonucleic acid (DNA) Ferrier 2014 6 DNA structure and properties  DNA is a macromolecule and its structure is hierarchical   Primary structure   Base pair interactions between separate chains to form helical structures Tertiary structure   Sequence and structure of nucleotides Secondary structure   Similar to the structure of proteins A, B, and Z-DNA conformations Quaternary structure  Interactions with histones and folding into chromosomes DNA structure and properties  The base composition of DNA determines its physical properties  Two chains of DNA combine to form a double helix  The chains are oriented in an anti-parallel orientation     One purine base always forms H-bonds with one pyrimidine base One strand is complementary to the other   i.e. one chain is oriented 5’ to 3’ and the other is oriented 3’ to 5’ The bases of one strand form hydrogen bonds (H-bonds) with the bases of the other strand Given one strand, the cell can synthesise the other The dexoyribosephosphate backbone is oriented on the ‘outside’ of the molecule and the bases are stacked on the inside of the molecule 7 Structure of the double helix of DNA If DNA were represented as a ladder the base pair spacing would be much larger than in a helix. A base in one chain is hydrogenbonded to the complementary base in the other chain: this is a ‘base pair’ or bp. There are about 3 billion base pairs in the human haploid genome! A helix compacts the entire structure. Note how base pairs are perpendicular to the axis of the helix and ‘stack’ on top of each other in the interior of the helix. Structure of the double helix of DNA The chains of DNA are anti-parallel to each other Note that a purine base (A/G) always forms H-bonds with a pyrimidine base (T/C) Ferrier 2014 8 DNA structure and properties  Base pairing occurs through formation of H-bonds  Watson-Crick base pairing  Adenine (A) always H-bonds (pairs) with thymine (T)   Guanine (G) always H-bonds with cytosine (C)   Two H-bonds formed and hence a ‘weaker’ link than the G-C pair Three H-bonds formed and hence a ‘stronger’ link than the A-T pair Chargaff’s rules state that in a sample of double stranded DNA (dsDNA):  The amount of guanine (G) equals the amount of cytosine (C)  The amount of adenine (A) equals the amount of thymine (T)  The amount of purines equals the amount of pyrimidines  The ratio (A+G)/(T+C) = 1 for all natural DNAs  This does not mean that G+C = A+T!  GC content (defined as (G+C)/(A+T+G+C) x 100%) varies between species DNA structure and properties  DNA double helix is stabilised by  H-bonds between base pairs  Hydrophobic interactions between base pairs  ϖ-ϖ interactions through base pair stacking   Attractive non-covalent interactions between the aromatic rings of the bases stacked parallel one above the other within the core of the DNA helix Interaction of the sugar-phosphate backbone with water (hydration) 9 DNA structure and properties  DNA helix can be denatured (unwound and strands separated) by disrupting Hbonds  pH  Heat   Melting temperature (Tm, temperature at which half of helical structure is lost)  DNA with high G-C pair content has a higher Tm than DNA with a high A-T pair content  More favourable stacking energy for G-C pairs than for A-T (or A-U pairs in RNA) because of the relative positions of chemical groups outside of the base ring.  The 3 H-bonds in the G-C pair vs the 2 H-bonds in the A-T pair do not in fact determine higher stability. Heat and pH affect H-bond interactions but do not affect phospho-diester bonds of the sugar-phosphate backbone Thermal stability of DNA structure Ferrier 2014 10 DNA can be renatured and hybridized  DNA can be renatured  When temperature or pH is returned to normal, the denatured, unwound segments of DNA can spontaneously rewind or anneal to give the helical structure   DNA denaturation/renaturation is part of the basis for the polymerase chain reaction (PCR) method! Nucleic acids from different species can form hybrids  If DNA from two species is completely denatured and then allowed to renature then hybrids consisting of one chain from one species and one chain from the other species may form  Extent of hybridization depends upon the sequence similarity between the species The double helix of DNA is flexible  DNA can bend, twist and turn  DNA can change conformation (it has conformational polymorphism)  Important for its function  Important for packaging, transcription, translation and recombination  Influences DNA-protein, DNA-DNA and DNA-RNA interactions 11 DNA double helix conformations A-conformation Right-handed Short and broad 11 bp per turn 23 Å diameter Found in dehydrated, non-physiological conditions B-conformation Right-handed Longer and thinner 10 bp per turn 20 Å diameter Usual in vivo conformation Z (zig-zag)-conformation Left-handed Longest and thinnest 12 bp per turn 18 Å diameter Found in G-C rich regions Nelson & Cox 2000 Ribonucleic acid (RNA)  RNA is mostly a single-stranded molecule  Can have double-stranded RNA  Shorter chain of nucleotides compared with DNA  RNA can form H-bonds through complementary base pairing   RNA contains ribose as sugar   Forms intra-strand double helices and other structures The hydroxyl group at the 2’ position of ribose make RNA less stable than DNA because it is prone to hydrolysis Many biological roles 12 RNA structure and properties  RNA is a macromolecule and its structure is hierarchical  Primary structure   Secondary structure   Base pair interactions within a single RNA chain to form various structures, such as stems and loops Tertiary structure   Sequence of nucleotides Three-dimensional structure of folded RNA Quaternary structure  Interactions of different RNA molecules in the ribosome Secondary and tertiary structure of RNA Example of secondary structure of RNA Examples of tertiary structure of RNA loops stems Messenger RNA Transfer RNA 13 Types of RNA  mRNA - messenger RNA   rRNA – ribosomal RNA     Non-coding RNA involved in gene expression snRNA - small nuclear RNA   Binds amino acids and transfers to ribosome during protein synthesis (translation) About 15% of total RNA content miRNA – micro RNA   Component of the ribosome, the organelle responsible for protein synthesis Most abundant of the RNA types: about 80% of total RNA content tRNA – transfer RNA   Transcribes the genetic code from DNA and then acts as template for the translation of the nucleic acid code to the protein ‘code’ Splicing of messenger RNA And multiple others… mRNA and DNA interaction Anti-parallel chains and complementary base pairs between a a single chain of DNA and a chain of mRNA   The ‘code’ from DNA is transcribed to the similar ‘code’ of RNA The mRNA then acts as template for the translation of the nucleic acid code to the protein code (sequence of amino acids) Ferrier 2014 14 tRNA – transfer of amino acids  There is at least one tRNA for each amino acid  The tRNA attaches the amino acid at the 3’ end  The tRNA-amino acid complex (sometimes called ‘charged’ tRNA) then interacts with the ribosome, where translation occurs  The anti-codon region of tRNA specifically interacts with the codon region of mRNA when building the protein sequence on the ribosome Ferrier 2014 rRNA – size diversity in the ribosome The size of rRNA is measured by the Svedberg (S) unit, which is determined by ultracentrifugation. Generally, the larger the S the larger the rRNA! rRNAs interact with proteins and each other as structural components of the ribosome Ferrier 2014 Ferrier 2014 15 DNA to mRNA to protein Nucleotides in other molecules Adenosine triphosphate (ATP) is an important molecule, mainly responsible for the mediation (transfer) of energy NH2 N O -O P O- O P O- N O O O P O N N O OH H H OH H OH Note: ATP has a ribose sugar 3D ‘Ball and Stick’ (Wikipedia) 16 Nucleotides in other molecules Nicotinamide adenine dinucleotide (NAD) is an enzyme co-factor involved in reduction-oxidation (redox) reactions Two nucleotides in the same molecule 3D ‘Ball and Stick’ (Wikipedia) Nucleotides in other molecules Coenzyme A (CoA) is an important activated molecule in metabolism (fatty acid synthesis and citric acid cycle) 3D ‘Ball and Stick’ (Wikipedia) 17 Nucleotides in other molecules Uridine diphosphate-glucose (UDP-glucose) is an ‘activated’ form of glucose and precursor in glycogen synthesis 3D stick representation (PubChem) Faculty of Health Sciences | School of Pharmacy and Biomedical Sciences Nucleotides in other molecules Cyclic adenosine monophosphate (cAMP) is an important intracellular signalling molecule 3D ‘Ball and Stick’ (Wikipedia) 18 Further reading  DNA, RNA and the Human Genome (online course from Western Oregon University): https://wou.edu/chemistry/courses/online-chemistry-textbooks/ch450-and-ch451biochemistry-defining-life-at-the-molecular-level/chapter-4-dna-rna-and-the-humangenome/  Structure of nucleic acids: https://www.youtube.com/watch?v=apaP9a079po  Nucleic acids - RNA and DNA structure: https://www.youtube.com/watch?v=7AtO8DuWsck  Ferrier, D.R. (2014) Biochemistry. 6th Edition. Parts of chapters 15-17. Lippincott Williams and Wilkins.  Nelson and Cox (2008) Lehninger’s Principles of Biochemistry. 5th Edition. Worth Publishers. 19