CHAPTER 3 NUCLEIC ACID TS. VÕ ĐÌNH LỆ TÂM ĐT: 0121 896 7699 E-mail: cntpsh@gmail.com CONTENT Nucleotide DNA, RNA Properties Biosynthesis of nucleotide Molecular biology in Food technology Genetic engineering GMO, GMF NUCLEIC ACID - HISTORY Isolated nuclein (from nuclei of white blood cells) nucleic acid Nucleic acids: polynucleotides Friedrich Miescher (1844-1895) Swiss physician and biologist NUCLEOTIDE STRUCTURE PHOSPHATE SUGAR (5C) NITROGEN BASE (C-N ring) GROUP (PO4) Ribose (RNA) PURINES PYRIMIDINES Deoxyribose (DNA) Adenine (A) Thymine (T); Uracil (U) Guanine(G) Cytocine (C) NUCLEOTIDE NUCLEOTIDE STRUCTURE - SUBUNITS NUCLEOTIDE STRUCTURE - BONDS NUCLEOTIDE STRUCTURE - PENTOSE POLYNUCLEOTIDE-BACKBONE THE SUGAR-PHOSPHATE BACKBONE Sugar-phosphate joined by phosphodiester bonds Sugar-phosphate in polynucleotide orientated in the same direction 3’-OH group of the sugar in one nucleotide forms an ester bond to the phosphate group on the 5’-carbon of the sugar of the next nucleotide POLYNUCLEOTIDE-BACKBONE NUCLEOTIDE STRUCTURE-BASE Bases Attached to the 1st Carbon of sugar NUCLEOSIDE & NUCLEOTIDE • Nucleoside – consists of a nitrogen base linked to C1’ of a ribose or deoxyribose (glycosidic bond) – Named • for purines: changing the nitrogen base ending: -ine to - osine : adenine adenosine • for pyrimidines: changing the nitrogen base ending (osine to – idine: cytosine cytidine NUCLEOSIDE & NUCLEOTIDE • Nucleotide – nucleoside forms a phosphate ester with the C5’-OH group of ribose or deoxyribose – Named: using the name of the nucleoside followed by 5’- monophosphate: adenosine 5’-monophosphate • Oligonucleotide: short nucleotide (<100 nt) (primers for DNA replication) NAMES OF NUCLEOSIDES AND NUCLEOTIDES ATP - MAJOR ENERGY SOURCE FOR CELLULAR ACTIVITY ATP: adenosine triphosphate All cells need chemical energy carried out by ATP Molecules in food store chemical energy in their bonds Starch molecule Glucose molecule HOW DOES ATP STORE ENERGY? Energy is stored in the last high energy phosphate bond (it can store more energy than other types of bonds) The cell stores energy by bonding a phosphate to ADP (Adenosine Diphosphate) ATP-CHEMICAL STRUCTURE + + HOW DOES CELL GET ENERGY FROM ATP?: HYDROLYSIS H2 O By breaking the high- energy bonds between the last two phosphates in ATP THE ADP-ATP CYCLE ATP ATP-ase Synthetase ATP & CHEMICAL ENERGY Organisms break down carbon-based molecules to produce ATP Carbohydrates: the most commonly broken down to make ATP – not stored in large amounts – up to 36-38 ATP from one glucose molecule Fats store the most energy: 80 percent of energy in the body – about 146 ATP produced from a triglyceride Proteins: the least likely to be broken down to make ATP – amino acids not usually needed for energy WHEN IS ATP MADE? During Cellular Respiration • Includes pathways that require oxygen • Glucose is oxidized and O2 is reduced • Glucose breakdown one molecule of glucose 36-38 ATP molecules TYPES OF NUCLEIC ACID DeoxyriboNucleic Acid (DNA) contains genetic information of an organism in the cell nucleus and mitochondria RiboNucleic Acid (RNA) throughout the cell, much more abundant than DNA assisting in the expression of DNA to protein DNA STRUCTURE – DISCOVERY X-ray diffraction photo of DNA (1952) by Rosalind E. Franklin Rosalind Elsie Franklin(1920-1958), Maurice Hugh Frederick Wilkins British biophysicist and X-ray (1916-2004), New Zealand- crystallographer, born English physicist Took X-ray diffraction photo of DNA and molecular biologist (1952) DNA STRUCTURE – DISCOVERY Francis Harry Compton Crick James Dewey Watson (1928) (1916-2004), American molecular biologist, English molecular biologist, geneticist, zoologist biophysicist, and neuroscientist Watson and Crick with their DNA model Francis Crick and James Watson working at the Cavendish Laboratory in Cambridge (1953), discovered the structure of DNA, a double helix DNA STRUCTURE – THE NOBEL PRIZE Crick, Watson and Wilkins won the Nobel Prize for medicine in 1962 Rosalind Franklin, Maurice Wilkins’s colleague, developed the technique to photograph a single strand of DNA died of cancer in 1958 could not be recognized in the Nobel Award WATSON & CRICK MODEL OF DNA Two strands of polynucleotides wind together run in opposite directions (antiparallel) Complementary WATSON & CRICK MODEL OF DNA Nucleotides bond between DNA strands H bonds Purine :: Pyrimidine pairing A :: T: 2 H bonds G ::: C: 3 H bonds Arranged in step-like pairs Determines the genetic information of DNA WATSON & CRICK MODEL OF DNA Ratio of A-T : G-C affects stability of DNA molecule Biotech procedures more G-C = need higher T° to separate strands BASE-PAIRING RULE OF NUCLEOTIDE Erwin Chargaff (1905-2002), Purine – Pyrimidine pairing Austrian American biochemist, A :: T (2 H bonds) discovered the base-pairing rule of G ::: C (3 H bonds) nucleic acids ERWIN CHARGAFF’S DNA DATA (1950-51) DNA - FUNCTION Storage of genetic information Self-duplication & inheritance Expression of the genetic message BIODIVERSITY Different arrangement of nucleotides in DNA biodiversity DNA - SEQUENCE Reading DNA sequence The sequence: read from 5’ to 3’ end using the letters of the bases • Ex: 5’—A—C—G—T—3’ Free 5’ end: phosphate group free 3’ end: - OH group DNA - THE DOUBLE HELIX Base pairs of two strands: consists of a purine and a pyrimidine the same width, keeping the two strands at equal distances from each other major groove 12 Å one helical turn minor 34 Å groove 6Å DNA - SUPERCOILS Each cell contains about 2 meters of DNA DNA “packaged” by coiling around a core of proteins (histones: rich in lysine and arginine residues) The DNA-histone: nucleosome In eukaryotic cells (animals, plants, fungi), DNA stored in the nucleus Properties of DNA Double stranded 1. Antiparallel strands DNA can be denatured 1.
Effect of G-C content on DNA denaturation Properties of DNA DNA can be renatured 1. Effect of time, concentration and complexity on renaturation General forms of a DNA helix 1. Z form FORMS OF A DNA HELIX DENATURATION & RENATURATION OF DNA Spectroscopy of Nucleic Acid UV absorption - Nucleic acids absorb UV light due to the aromatic bases - The wavelength of maximum absorption by both DNA and RNA is 260 nm (lmax = 260 nm) - Applications: detection, quantitation, assessment of purity (A260/A280) DIFFERENCES BETWEEN RNA & DNA RNA (ribonucleic acid) Differences between RNA and DNA - Pentose sugar: ribose, [deoxyribose in DNA] - Uracil replaces thymine - Single stranded [DNA: double stranded] - Much smaller than DNA - Three main types of RNA: ribosomal (rRNA), messenger (mRNA) and transfer (tRNA) RNA STRUCTURE TYPES OF RNA RIBOSOMAL RNA (rRNA) & MESSENGER RNA (mRNA) Ribosomal RNA 65% of Ribosomes ( Sites of protein synthesis) Messenger RNA Carries the genetic code to ribosomes Complementary to the DNA of the gene) TRANSFER RNA (tRNA) Transfer RNA Translates the genetic code from the mRNA Brings specific amino acids to the ribosome for protein synthesis Each amino acid is recognized by one or more specific tRNAs - one end: attaches to the amino acid - the other end binds to the mRNA (complimentary sequence) GENETIC CODE GENETIC CODE ORGANIZATION Single-base changes (single-nucleotide polymorphism) in the third position in a codon produce the same amino acid The second base specifies if the amino acid is polar or apolar (hydrophobic) Changes elsewhere in the codon produce a different amino acid, but with the same physical-chemical properties Polar R groups make the amino acid hydrophilic Non-polar R groups make the amino acid hydrophobic Ionic R groups make the amino acid hydrophilic READING THE GENETIC CODE Ex: determine the amino acid sequence coded by a section of a mRNA 5’—CCU —AGC—GGA—CUU—3’ According to the genetic code amino acids sequence CCU = Proline AGC = Serine GGA = Glycine CUU = Leucine mRNA section codes for the amino acid sequence Pro—Ser—Gly—Leu THE CENTRAL DOGMA OF MOLECULAR BIOLOGY (BY F. CRICK) Francis Harry Compton Crick (1916-2004) Replication: DNA is copied with very high fidelity Transcription: DNA genetic code is read and transferred to messenger RNA (mRNA) Translation: genetic code is converted to a protein