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BIOCHEMISTRY Textbook: “A Text Book of Biochemistry”, by Zhao Baochang, etc, 2004. Books for reference: 1. “Biochemistry”, by L. Stryer, 6th edition, W.H. Freeman and Company, 2006. 2. “Instant Notes in Biochemistry”, by B.D. Hames & N.M. Hooper, 2nd edition, BIOS Scientific Publishers Limited, 2000. Important Concepts 1. Biochemistry is the study of the molecular composition of living cells, the chemical reactions of biological compounds, and the regulation of these reactions. 2. Major components in body include water (55%), protein (19%), fat (19%), inorganic matter (7%), carbohydrate (<1%), and nucleic acid (<1%). 3. Metabolism refers to all the chemical reactions of a living organism. Why to study? 1. Biochemistry is one of the basic courses that can help you to understand the physiological and pathological processes in the body at molecular levels, and more importantly, to use the knowledge to . 2. Biochemistry is also a powerful tool in life-scientific studies—prepares you to be a good scientist. How to study? 1. Classroom study: it is impossible for a lecturer to give all details of the knowledge in a limited lecturing-time, but it is important for the students to catch the main points during the class. 2. Your study should not be limited to classroom and textbook, but be anyway that helps you understand well the concepts and the principles of biochemistry, such as discussions between teacher-students and among students, lab study, scientific journals... Chapter 1. Structures and Functions of Nucleic Acids Nucleic Acids include deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). The genes of all cells and many viruses are made up of DNA, while RNA serves as the machinery of protein synthesis. The flow of genetic information: DNA transcription translation RNA Protein 1. Composition of nucleic acids 1) Phosphate DNA: Adenine(A), Guanine(G), Thymine(T), Cytosine(C) 2) Bases RNA: Adenine(A), Guanine(G), Uracil(U), Cytosine(C) DNA: deoxyribose 3) Pentoses RNA: ribose Structures of bases Purine Guanine(G) Pyrimidine Cytosine(C) uracil(U) Adenine(A) Thymine(T) Structures of pentoses Deoxyribose ribose 2. Nucleosides and nucleotides 1) Nucleoside: base-pentose Deoxyadenosine Adenosine 2) Nucleotide: base-pentose-phosphate O Deoxyadenosine monophosphate (dAMP) Adenosine monophosphate (AMP) 3) Common nucleotides: A) Deoxyribonucleotides dAMP dADP dATP B) Ribonucleotides HO AMP ADP ATP Names of nucleoside and nucleotides Base In RNA: adenine guanine cytosine uracil In DNA: adenine guanine cytosine thymine Ribonucleoside Ribonucleotide Adenosine Guanosine Cytidine Uridine Adenosine-5’-monophosphate Guanosine -5’-monophosphate Cytidine -5’-monophosphate Uridine -5’-monophosphate deoxyadenosine deoxyguanosine deoxycytidine deoxythymidine deoxyadenosine-5’-monophosphate deoxyguanosine-5’-monophosphate deoxycytidine-5’-monophosphate deoxythymidine-5’-monophosphate Abbreviated names of nucleoside mono-, di-, tri- phosphates Base NMP Ribonucleotides: NDP NTP A G C U ADP GDP CDP UDP ATP GTP CTP UTP dADP dGDP dCDP dTDP dATP dGTP dCTP dTTP AMP GMP CMP UMP Deoxyribonucleotides: A G C T dAMP dGMP dCMP dTMP Ultraviolet absorption spectra of ribonucleotides Ultraviolet absorption of nucleotides is due to the optical property of the bases. The wavelength at 260nm is often used to quantitatively analyze bases, nucleosides, nucleotides, or nucleic acids. 3. Primary structure of nucleic acids 1) Nucleotides are linked by 3’,5’phosphodiester bonds to form oligo- or poly- nucleotides. RNA: polynucleotide chains DNA: polydeoxynucleotide chains 3’,5’- phosphodiesters - O 5’- end - O P O O CH2 Base O H H O H H 3’,5’- phosphodiesters - O P O O CH2 Base O H H O H H - O P O O CH2 3’- end H Base O H H OH H Direction: 5’ 3’ 2) Primary structure of nucleic acids refers to the nucleotide sequence of the polynucleotide chain. The primary structure of a DNA chain may be expressed as: A 5’ P C P T P G P C P Or: 5’ pApCpTpGpCpT 3’ Or: 5’ ACTGCT 3’ T P OH 3’ 4. Stereo structures of DNA 1) The secondary structure of DNA Watson-Crick model: DNA double helix. • The two polynucleotide chains are coiled around a common axis in opposite directions. • The bases are on the inside of the helix, forming hydrogen bonds between the two chains by A-T and G-C complementary pairing. The phosphate and deoxyribose are on the outside as the backbones. The base sequence carries the genetic information. • Minor groove Major groove The DNA double helix Double helical structure of DNA Minor groove Major groove 34Å The DNA base pairs 2) The higher-level structures of DNA • Prokaryotic DNA: is circular double stranded and may be further folded into loops or supercoils with or without DNA binding proteins. • Eukaryotic DNA: is complexed with a histone octamer to form a nucleosome. Histones Five main types of histones: H1, H2a, H2b, H3 and H4. They are basic DNA-binding proteins. The histone octamer consists of 8 histones: two molecules of each H2a, H2b, H3 and H4, serving as a core of nucleosome. Prokaryotic DNA loops Structure of nucleosome Formation of chromosome About DNA DNA is of paramount importance for storing, expressing and transmitting genetic information. Growth, reproduction and hereditary characteristics depend on DNA. DNA contains the information that directs the development of an organism. DNA is able to replicate each time a cell divides and also have the information that is to be selectively expressed. 5. RNA Structure • Most RNA molecules are single-stranded polymer chains consisting of ribonucleotides linked by 3’5’ phosphodiester bonds. However, some regions of RNA can form double-stranded structures by A-U and G-C base pairing within the single chain itself. RNA and DNA structures RNAs in the mammalian cell RNA rRNA mRNA tRNA HnRNA Function component of ribosome template for Pr. Synthesis transporter of amino acids precursor of mRNA ribosomal RNA messenger RNA transfer RNA heterogeneous nuclear RNA SnRNA small nuclear RNA splicing of HnRNA SnoRNA small nucleolar RNA processing of rRNA ScRNA small cytoplasmic RNA signal-peptide recognition 1) Structure of mRNA The structural characteristics of mRNA: a) A cap structure at the 5’ end: protects the 5’ end from degradation by nuclease and helps in the initiation of protein synthesis. b) A polyA tail at the 3’ end: is not encoded by DNA but added after transcription. The polyA tail protects the 3’end from nuclease digestion and stabilizes the mRNA. c) A coding sequence at the center: encodes the amino acid sequence of a polypeptide. One mRNA only encodes one polypeptide chain in mammalian cell, but may encode several polypeptides in bacteria. Cap Non-coding Coding sequence Non-coding polyA tail 5’ 3’ The cap structure of mRNA 2) Structure of tRNA a) Secondary structure of tRNA: a cloverleaf structure containing an anticodon arm, a DHU arm, a TC arm, and an amino acid acceptor stem. b) Tertiary structure of tRNA: at the level of secondary structure the molecule further folds to form a “L” shape 3-d structure. Secondary structure of tRNA Tertiary structure of tRNA 3) Structure of rRNA A ribosome consists of a small and a large subunit, each of which contains proteins and rRNAs forming a site for protein synthesis. • Types of rRNA: prokaryotes eukaryotes Small subunit 30S 40S rRNA 16S 18S Large subunit 50S 60S rRNA 23S, 5S 28S, 5.8S, 5S Types of rRNA b) Secondary structure of rRNA: complex, different in size, composition, and 3-d structure. About RNA • mRNA participates in the process of selective expression of genetic information stored in DNA. • tRNA serves as carrier of genetic information to the site of protein synthesis. • rRNA is an essential component of ribosomes. 6. Properties of nucleic acids 1) Denaturation and renaturation Denaturation: due to the action of some physical (heat etc.) or chemical (organic solvents etc.) factors the native structure of a nucleic acid molecule can be changed, resulting in loss of its biological functions and showing several physical changes (increase in viscosity and in absorbance of UV light). Renaturation: when the denaturing factors are removed, the denatured nucleic acid molecules may restore their native structures with recovery of their biological functions and physical properties. Melting temperature (Tm) of DNA: the temperature at which 50% of the maximum optical density is reached. Relative optical density (260nm) Melting curve 1.4 1.3 1.2 1.1 Tm 1.0 30 50 70 Temperature (oC) 90 2) Hybridization: a process of association through base-pairing between two polynucleotide chains that are complementary in base sequence to each other. Hybridization can occur between DNADNA, RNA-RNA, or DNA-RNA polynucleotide chains of different origins. Hybridization is a powerful technique that can be used for probing specific genes. Principles of nucleic acid hybridization