Introduction to Molecular Biology 1st Course PDF

Introduction to molecular Biology Pr. Raouf Alami CM1 Module : cell biology and molecular biology...

Introduction to molecular Biology Pr. Raouf Alami CM1 Module : cell biology and molecular biology Molecular biology Année Universitaire: 2022-2023 www.um6ss.ma Course contents 1. Objective of this course 2. Central Dogma of molecular biology 3. How DNA was discovered? 4. DNA molecular composition 1. Deoxyribose 2. Phosphate 3. Nitrogenous bases 5. How DNA is built 6. Ester bonds and the making of the DNA backbone 7. how the double helix is stabilized? 8. Physical and Chemical Properties of DNA 1. Solubility 2. Absorption 3. Denaturation and Renaturation 4. DNA electrical charge 5. DNA packaging 9. Mitochondrial DNA 10. Chromosomal DNA vs Mitochondrial DNA 11. Bacterial DNA 12. DNA in Eukaryotes and Prokayotes Course Part 2 : RNA 2 1 Learning objectives and course organization In this lecture we will present some important dates in molecular biology. We will also describe the structure, the composition the and characteristics of DNA and RNA 3  Introduction The field of molecular biology study macromolecules and the macromolecular mechanisms found in living things. Molecular biologist study how molecules interact with one another in living organisms to perform the functions of life. Such as The molecular nature of the gene Mechanisms of gene replication, Mutation Gene expression  Central dogma of molecular biology Is a theory stating that genetic information flows only in one direction, from DNA, to RNA, to protein, or RNA directly to protein. What is a Dogma? This is something held as an established opinion That is set by the members of a group without being questioned or doubted  Where molecular biology come from? Molecular biology originated in the 1930s and 1940s, The emergence of molecular biology represented a convergence of work by: On a common problem: the nature of inheritance. How was DNA discovered?  DNA was first identified in the late 1860s by Swiss chemist Friedrich Miescher (He called nuclein) The term “molecular biology” was introduced in 1938 by Warren Weaver, director of the Natural Sciences section of the Rockefeller Foundation And the field picked up momentum in the 1950s and 1960s What event in history made a breakthrough in molecular biology? 1950, Erwin Chargaff found that in DNA, the ratios of adenine (A) to thymine (T) and guanine (G) to cytosine (C) are equal. In 1953, the double helical structure of the DNA molecule was discovered by James Watson and Francis Crick 1961 – the messenger RNA was discovered by Francois Jacob and Jacques Monod ( the existence of an intermediary between DNA and its protein products) 1961, Crick et al. published a paper where they deciphered the triplet codon pattern. The central dogma DNA Transcription Decoded by tRNA mRNA Translation Proteins + rRNA Ribosomes Protein DNA Deoxyribonucleic acid Double Stranded Nucleic Acid Deoxy sugar  The nitrogen bases were discovered: uracil in the early 20th century In living organisms there are 4 deoxyribonucleic acids that vary in their nitrogenous bases adenine, cytosine, guanine, thymine, and Dr. Phoebus Levene  Abundance of the nucleiotides in differents organismes Once the primary structure that creates the DNA molecule was known, How these nucleotides are arranged to create DNA secondary structure? The DNA secondary structure was the most important biological discoveries in the 20th century One of the first clues came from 1949 , when Erwin Chargaff compared the relative abundance of the nucleiotides in differents organismes Erwin Chargaff discovery was confirmed accross many species Source of Adenine % Thymine % Guanine % Cytosine % DNA Human 30 30 20 20 Rat 28 28 22 22 Yeast 31 31 19 19 Turtle 28 28 22 22 Salmon 29 29 21 21 Sea 33 33 17 17 urchin Erwin Chargaff found that in DNA, the ratios of adenine (A) to thymine (T) and guanine (G) to cytosine (C) are equal. Based on Chargaff discovery, James Watson and Francis Crick strongly support base pairing evidence in DNA And suggested that DNA was most likely double stranded  Rosaland Franklin &Maurice wilkins Rosaland Franklin Maurice wilkins X Ray Chromatography X-ray diffraction Three conclusions : image of DNA 1. DNA has a consistant width 2. There is a repeating pattern within DNA 3. DNA must be helical  In collaboration with Wlikins and Franklin, Watson and Crisk used a measurment to define the geometry of the components of the DNA. Watson and Crick Using Chargaff rule (A=T and C=G) By arranging base pairing nucleotides : C- G and T-A, side by side, and strands runing in the opposite directions, all the discoveries could be explained  How the double helix is stabilized? The geometry of CG or GC and for AT or TA is similar allowing symetry and base stacking. It mostlty has to do with the distance with the backbones Always purine facing a pyrimidine  hydrogen bonds Note : Noncovalent bonding holds the two strands of the DNA double helix together  Watson and Crick findings Anti parallel Watson and Crick suggested that the DNA is composed with double strands running in the opposite directions 5’ 3’ The nucleic backbone is composed with alternating phosphate and sugar P-S-P-S- P-S, ……….. With 1. One phosphate always on the 5’ at the end of the first strand 2. One deoxyribose at the 3’ end of the other strand The strands twist to form a double helix. A spiral with 2 nucleic backbones running on the opposite direction with the nitrogenous bases facing inward 3’ 5’  The Nobel prize The Nobel Prize in Physiology/Medicine in 1962 was awarded to James Watson, Francis Crick and Maurice Wilkins for their discovery of the molecular structure of DNA Rosaland Franklin didn’t get the nobel prize because she passed away. (1958) In 2014, Watson sold his DNA co-discoverer’s Nobel Prize medal for record US$4.7 million at auction  Central Dogma of molecular biology The central dogma of molecular biology is a theory stating that genetic information flows only in one direction, from DNA, to RNA, to protein, or RNA directly to protein. DNA → RNA → Protein RNA to Protein This was what Crick meant when he said that once : information had gone from DNA into the protein, it could not get out of the protein and go back into the genetic code. DEOXYRIBONUCLEIC ACID Stores and passes on genetic information from one generation to another. The DNA is shaped like spiral staircase or twisted ladder Rails Spreaders  DNA composition DNA is made of 3 molecules: 1. Deoxyribose 2. Phosphate 3. Nitrogenous bases 1- Sugar Ribose is a sugar like glucose, but with only 5 carbon atoms in its molecule. Deoxyribose is almost the same but lacks 1 oxygen atom. Both molecules may be represented by the symbol: Pentagon Hexagon Ribose/deoxyribose Glucose Ribose Ribose is a single-ring pentose [5-Carbon] sugar. Ribose is found in the RNA 5’ The numbering of the carbon atoms runs clockwise 1 4’ 1’ 3’ 2’ The carbon numbering is the key for describing DNA directionality Deoxyribose Is a ribose but missing the hydroxyl (-OH) group at the carbon 2’ Note the absence of the hydroxyl (- OH) group on the 2’ carbon in the deoxy-ribose sugar Deoxyribose is found in the DNA Ribose vs. Deoxyribose  2- Phosphate molecule Phosphate, chemical formula PO43-, is a chemical compound made up of one phosphorus and four oxygen atoms. When it is attached to a molecule containing carbon, it is called a phosphate group In the nucleic acid (DNA or RNA) the phosphate group is attached to the sugar 5’ molecule on the 5' carbon. The DNA backbone  How the DNA backbone is made? The outer parts of the of the ladder are made of : Deoxyribose + phosphate These are joined together by ester bonds or phosphodiester bonds Ester bonds Phosphodiester bonds are central to all life on Earth, as they make up the backbone of the strands of nucleic acid  Forming the DNA backbone The hydroxyl groups on the 5'- and 3'- carbons link to the phosphate groups to form the DNA backbone. 5’ 3’ new water molecule + H2O Joined together by ester bonds 5’ 3’ 3’ phosphodiester bonds the circled red H-OH forming a new water molecule that gets lost. ESTER BONDS make the ladder’s rails These strands are also sometimes called Watson and Crick Crick strand Watson strand What makes the ladder’s steps? The steps of the ladder are made of pairs of nitrogenous bases.  The bases For DNA, the bases are :. Cytosine (C) Thymine (T), Adenine (A) Guanine (G)  3- Attaching a base and making a nucleotide The final piece that we need to add to this structure before we can build a DNA strand is one of four organic bases. These bases attach in place of the -OH group on the 1' carbon atom in the sugar ring. 1’ What we have produced is known as a nucleotide. The nitrogenous bases Pyrines double ring structure Pyrimidines single ring structure How do you remember Purines from Pyrimidines? Pyramids Pyramids (like pyrimidines) are "sharp" and thus they CUT, Cytosine, Uracil and Thymine.  Summary A nucleotide is the basic building block of nucleic acids (RNA and DNA) Nucleoside versus nucleotide Hydrogen bonds Stabilize the structures of DNA Adenineine pairs preferentialy with thymine through 2 hydrogen bonds Guanine pairs preferentialy with Cytosine through 3 hydrigen bonds Each base forms multiple hydrogen bonds with its complementary base on the opposite strand  DNA 3D structure The double helix of the DNA is very regular and forms two repeating and alternating structures: the minor groove and the major groove A twist that repeats every 3.4–3.6 nm 2 nanometres wide Deep and wide Narrow and shallow The major groove is deep and wide, the minor groove is narrow and shallow.  DNA 3D structure How many base pair per turn? The structure of the DNA is very regular, each turn of the helix measure 10 base pair 0.7 nanometres DNA 3D structure 3 possible forms of DNA A B Z Right-handed Right-handed Left-handed double helix double helix double helix The most common DNA conformation and is the B form Physical and Chemical Properties of DNA 1- Solubility DNA is soluble in water. Its highly charged phosphate-sugar backbone gives it its polarity. However, in the presence of salt and alcohol, DNA is insoluble. he hydrophilic nature of DNA is due to the presence of phosphate groups  Physical and Chemical Properties of DNA 2-Absorption DNA bases can absorb ultraviolet light. At 260 nanometers A spectrophotometer can measure this absorption. DNA A double-stranded DNA absorbs 1.00 unit at 260 nm.  Physical and Chemical Properties of DNA 3- Denaturation and Renaturation DNA is a highly stable molecule. In the process of denaturation, an unwinding of DNA double-strand takes place, resulting in two separate single strands on applying high temperature, extreme pH, etc. Separate single strands rewind on cooling and the process is known as renaturation On heating, both strands denature, and on cooling, they can renature  Physical and Chemical Properties of DNA 3- Denaturation and Renaturation Denaturation and Renaturation the region of higher concentration of C-G has a higher melting temperature because these bases are bonded with three hydrogen bonds, which require more energy to break. A-T, which is only bonded with two hydrogen bonds requires less energy to break oppen The melting temperature varies on the DNA sequence  Physical and Chemical Properties of DNA 3- Denaturation and Renaturation Melting temperature  Physical and Chemical Properties of DNA 4- DNA electrical charge DNA is negatively charged The phosphate group of DNA is negative. The overall charge of DNA is thus negative.  Physical and Chemical Properties of DNA 4- DNA packaging ∼10 μm in diameter and contains 46 chromosomes consisting of ∼2 m of DNA Chromosomal DNA is packaged inside microscopic nuclei with the help of histones. DNA packaged around histones Complexes DNA+histones = nucleosomes Mitochondrial DNA Mitochondria are the major source of ATP in the cell. ~16 kb circular molecule composed of double-stranded DNA Mitochondrial DNA contains 37 genes : 2 encode for rRNAs, 22 for tRNAs and mRNAs, and 13 proteins used for the oxidative process Mitochondrial DNA is a circular chromosome found inside mitochondria. Each human cell contains approximately 100 mitochondria and each mitochondrion has 2 to 10 copies of mtDNA The mtDNA replicates independently. It replicates and translates to produce their specific protein.  Mitochondria and chloroplasts Mitochondria are the "powerhouses" of the cell, breaking down fuel molecules and capturing energy in cellular respiration. Chloroplasts are found in plants and algae. They're responsible for capturing light energy to make sugars in photosynthesis.  Chromosomal DNA vs Mitochondrial DNA Feature Nuclear DNA Mitochondrial DNA Size 3 x 10 (9) 15 569 shape Linear double helix Circular double helix inhéritance Both parents Maternal Nulber of 20 000 protein 13 protein- genes coding coding +24 non-protein, coding intron Found in almost absent every gene % of coding 3% 93% DNA histones Associated with the Not associated DNA with DNA Bacterial DNA The DNA of bacterial cells is found loose in the cytoplasm. It is called chromosomal DNA and is not contained within a nucleus. A plasmid is a small, extrachromosomal DNA molecule within a cell that is physically separated from chromosomal DNA and can replicate independently.  Bacterial DNA Circular DNA seen under an electron microscope.  DNA in Eukaryotes and Prokayotes Eukaryote Prokaryotes DNA in the nucleus DNA in the cytoplasm DNA bound to protein (histones) DNA naked DNA is lenear DNA circular Unusally has introns Usually no intron Eukaryotic cells contain mitochondrial circular DNA in addition to smaller, DNA in addition to nuclear DNA. transferable DNA plasmids. Human DNA mt DNA vs bacterial DNA  Quiz Quiz 1 - MB is a convergence of many disciplines such as : Geneticists Chemists Physicists  Quiz 2- What was the commun problem that gathers all this disciplines? Looking for nature of inheritance  Quiz 3- What are the differents nitrogenous bases in the DNA? 4-What was the Charfaff’s rule? C=G and A=T 5-What kind of technic Rosaland Franklin and Maurice Wilkins used to see the X-ray diffraction image of the DNA? X Ray Chromatography  Quiz 6- What was Rosaland Franklin and Maurice Wilkins 3 conclusions? 1. DNA has a consistant width 2. There is a repeating pattern within DNA 3. DNA must be helical 7- Whats is the most important difference between the purine and the purimidine? Purine : 2 rings Pyrimidine : 1 ring 8- What are the pyrimidine bases? CUT  Quiz 9- What is the findings of W and C? The nucleic backbone is composed with alternating phosphate and sugar P-S-P-S-P-S, ……….. The strands twist to form a double helix. A spiral with 2 nucleic backbones running on the opposite direction with the nitrogenous bases facing inward 10 - What is the difference between Glucose and Ribose? Pentagon/hexagon 11- What is the number of the deoxycarbon in the sugar 2’ 12- What are the sugars that attach to the Phospate molecule? 3’ and 5’ 13- What is a phosphate group? When the phosphate molecule is attached to Quiz a molecule containing carbon, it is called a phosphate group 14- Why Phosphodiester bonds are important for life on Earth? Making the backbone of DNA 15- What carbon atom is not involved in the formation of the DNA backbone? 2 and 4 16- What is the direction of the strand called watson strand? 5’ to 3’ 17- What is the diference between Nucleoside and Nucleotide? Nucleoside does’t have phosphate Nucleoside =nucleotide - phosphate  Quiz What is the central dogma of molecular biology? information goes from DNA into the protein, it could not get out of the protein and go back into the genetic code. Physical and Chemical Properties of DNA 1- What is the width of the DNA? 2 nanometers Quiz 2- How much is the DNA twist? 3.4–3.6 nm 3- What is the differene between the major and the minor groove? Major : Deep and wide Minor : narrow and shallow 4- DNA hydrophobic or hydrophilic? Hydrophilic because of the P group What makes DNA chargd negatively? Phosphate groupe charged neg What wavelength of light is absorbed by DNA? 260nm It separates the double strands What happens during the denaturation of DNA? What is the melting temperature? Half of the DNA molecule are denatured How the 46 chromosomes can fit inside a nucleus of Packaged with histones 10µM