分子生物学157744381

Molecular Biology大连理工大学What we learned last class Teaching arrangement of this course Definition of Molecular Biology Goals of Molecular Biology Major events in the history of MB Preview of some concepts and processes of Molecular BiologyMolecular Biology大连理工大学Translation Process of generating a protein or polypeptide from an mRNA molecule is known as translation. Protein: a polymer or chain of amino acids, whose sequence is determined by the mRNA template 3 nulceotides code for 20 naturally occurring amino acids 43 = 64; thus several trinucleotide sequences (codons) correspond to a single amino acid. There is no nucleotide between codons, and a few codons represent start and stop. Molecular Biology大连理工大学Molecular Biology大连理工大学Molecular Biology大连理工大学Molecular Biology大连理工大学Molecular Biology大连理工大学Molecular Biology大连理工大学Protein StructureMolecular Biology大连理工大学Biological function of proteinsEnzyme catalysis: DNA polymerases, lactate dehydrogenase, trypsinTransport: hemoglobin, membrane transporters, serum albuminStorage: ovalbumin, egg-white protein, ferritinMotion: myosin, actin, tubulin, flagellar proteinsStructural and mechanical support: collagen, elastin, keratin, viral coat proteinsDefense: antibodies, complement factors, blood clotting factors, protease inhibitorsSignal transduction: receptors, ion channels, rhodopsin, G proteins, signalling cascade proteinsControl of growth, differentiation and metabolism: repressor proteins, growth factors, cytokines, bone morphogenic proteins, peptide hormones, cell adhesion proteinsToxins: snake venoms, cholera toxinMolecular Biology大连理工大学Molecular Biology大连理工大学Molecular Biology大连理工大学Molecular Biology大连理工大学Molecular Biology大连理工大学Molecular Biology大连理工大学Molecular Biology大连理工大学Model Biological Systems Fundamental problems are solved in the simplest and most accessible system in which the problem can be addressed. These organisms are called model organisms. Virus 病毒 Bacteria 细菌 Bacteriophage 噬菌体 Yeasts 酵母 Animal cells and embryos 动物细胞和胚 Plant cells 植物细胞Molecular Biology大连理工大学Animal cells and embryos The nematode（线虫） Caenorhabditis elegans The fruit fly（果蝇） Drosophila melanogaster Silk worm The house mouse Mus musculus Mammalian Cell lines Embryonic stem cellsMolecular Biology大连理工大学Features of Model Systems The availability of powerful tools of traditional and molecular genetics. The study of each model system attracted a critical mass of investigators. (Ideas, methods, tools and strains could be shared) It depends on what question is being asked When studying fundamental issues of molecular biology, simpler unicellular organisms or viruses are convenient. For developmental questions, more complicated organisms should be used.Molecular Biology大连理工大学Features of Ideal Model Systems Shorter generation time Smaller amount of genetic material survives and breeds well in the lab many aspects of its phenotype (appearance)Molecular Biology大连理工大学The scientific processobservationhypothesispredictionexperimentsupportrefuteNew hypothesisMolecular Biology大连理工大学Rapid progress in molecular biology Rapid progress in molecular biology has generated literally thousands of volumes of detailed information about the structure and function of cellular components. Organizing those details in a comprehensive way represents a challenge for both the beginning student and the accomplished professional scientist. In order to develop a working knowledge of the discipline, details about molecular structure and function require attention, and generalizations need to be made. Molecular Biology大连理工大学Putting the details of molecular biology in perspective “Reductionist” approach believe the whole (organism) can only be fully understood when it is dissected and analyzed. Eventually, the lowest common denominators, such as the nature of the bonding between DNA and proteins, are revealed and general theories are constructed.Molecular Biology大连理工大学Putting the details of molecular biology in perspective “Holistic(整体整体)” approach emphasizes the integration of various biological phenomena into higher order systems. In some cases, those systems are represented by individual types of organisms, while in other cases several organisms are integrated into even higher orders of organization, such as the disciplines of animal or plant ecology.Molecular Biology大连理工大学Putting the details of molecular biology in perspective Individual molecules are, of course, the basis of all living activities. Nevertheless, in order to fully comprehend the function and significance of any single molecule, an excursion beyond the test tube and perhaps even out of the laboratory and on to biological field stations will eventually be necessary.Molecular Biology大连理工大学Rewards from Studying Molecular Biology First, the use of the “layering approach” to constructing a knowledge base, provides a learning tool that, once mastered, can be applied to other disciplines, ranging from poetry to physics. Second, studying molecular biology provides an excellent opportunity for enhancing ones analytical thought processes (so-called “critical thinking skills”). From time to time, experimental data are encountered. Learning how to interpret it usually involves analyzing entries in tables or graphs, and thereby improves problem-solving skills. Molecular Biology大连理工大学Rewards from Studying Molecular Biology Third, by learning the concepts and details of molecular biology, the beginning biology student is provided a gateway to virtually all other disciplines in biology, ranging from cell biology to genetics to population biology. Finally, should you, the beginning student, develop an interest in molecular biology, you might consider choosing a career in a field related to this discipline. Molecular Biology大连理工大学Major Aspects of Chapter 1 Concept of Molecular Biology Goals of Molecular Biology Major events in the history of MB Preview of Molecular Biology Model Biological Systems Lab Methods for Molecular Biology Scientific process Rewards from studying MBMolecular Biology大连理工大学Important concepts and features you are required to understandgenomeFunction of cells Prokaryotic and Eukaryotic cellsEukaryotic DNA Prokaryotic DNA4 phases of Eukaryotic Cell Cycle Central Dogma Chromosomehuman cell Chromosomegene locusfeatures of Structure of Nucleic Acidhydrogen bonds between basesG+C content and melting temperatureChemical and structural Difference between DNA and RNAchromatinnucleosomesforms of DNAenzymes involved in DNA Replicationcharacteristics of Mitochondrial DNAlevels of Gene expression why and how to study Gene expressiontranscriptionDifference between DNA Replication and transcriptionGenetic codes and codonsStop codonsDifferent types of RNA and functionsOpen Reading Frame (ORF)Promoter regionsmRNA untranslated regionsCoding and non-coding RNAexons and introns splicingtranslation and ribosomespolysomesFour levels of a proteins structureTypes of control in EukaryotesMutationCommon Tools in Molecular BiologyMajor applications of Molecular Biology Model Biological Systems and their featuresThe end of chapter 1Molecular Biology大连理工大学Chapter 2 The Macromolecules of the Cell use these four major classes of biological molecules.Molecular Biology大连理工大学ObjectivesThe nature of the components of the macromolecules(大分子） of the cell, and the chemical linkages by which they are joined to form biopolymers (生物多聚体)The role that non-covalent (非共价的)interactions play in determining the three-dimensional structure of macromolecules.Major lab methods for isolation and characterization of protein and nucleic acidsMolecular Biology大连理工大学Small Molecules vs Macromolecules Small molecules：molecular weights usually do not exceed several hundred. Inorganic ions Organic compounds Macromolecules: massive polymers, molecular weights over ten thousand Proteins Nucleic acids Polysaccharides (多糖多糖) LipidsMolecular Biology大连理工大学Monomers, Polymers and Macromolecules Biological macromolecules are formed by linking together a set of building blocks (monomers，单体) into long chains (a polymer). A massive polymer makes a macromolecule. Molecular Biology大连理工大学Condensation Reaction These monomers are linked together by a process called dehydration （脱水）脱水）synthesis (also called a condensation reaction) in which a covalent bond is formed between the two monomers while a water molecule is also formed from the OH groups. This reaction is catalyzed by enzymes. This same type of condensation reaction can occur to form different macromolecules, including proteins, nucleic acids, polysacchrides and lipids.Molecular Biology大连理工大学30 Most Common Monomers in CellsMoleculeNo. PresentNames of MoleculesRole in cellAmino acid 20Ala (A) Leu (L) Arg (R) Lys (K) Asp (N) Met (M) Asp (D) Phe (F) Cys (C) Pro (P) Gln (Q) Ser (S) Glu (E) Thr (T) Gly (G) Trp (W) His (H) Tyr (Y)Ile (I) Val (V)Monomers of proteinsnucleotides5AdenineThymine CytosineUracilGuanineMonomers for nucleic acidsSugars2RiboseComponent of nucleic acidsGlucoseComponent of polysaccharidesEnergy metabolismLipids3Choline胆碱 Components of phospholipidsGlycerolPalmitate棕榈酸 Molecular Biology大连理工大学ProteinsMolecular Biology大连理工大学Proteins are Linear Chains of Linked Amino AcidsException: proline only hasan imino groupMolecular Biology大连理工大学A Common Thread but a Unique IdentityMolecular Biology大连理工大学Amino acids 60 kinds in a cell, but only 20 involved in protein synthesis The 20 essential amino acids can be categorized into three groups Non-polar (hydrophobic)（疏水） Polar uncharged (hydrophilic)（亲水） Polar charged (hydrophilic)Molecular Biology大连理工大学Molecular Biology大连理工大学 Form salt bridgesContain groups that form hydrogen bonds with waterMolecular Biology大连理工大学Polar uncharged Cysteine Cysteine (Cys, C，半胱氨酸) has a thiol (硫醇) group (-SH)，which is often oxidizes（氧化）氧化） to cystine（光氨酸）.Disulfide bondMolecular Biology大连理工大学Amino acids- aromatic (芳香族，芳香族，3) Accounts for most of UV absorbance of proteins at 280 nmMolecular Biology大连理工大学Chirality 手性手性 -carbon is chiral (asymmetric 不对称的) except in glycine (甘氨酸，R is H) The two asymmetric forms are referred to as D and L D comes from dextrorotatory(右旋，right turning) L comes from levorotatory(左旋，left turning) All biologically active amino acids are of L form. Molecular Biology大连理工大学Molecular Biology大连理工大学Peptide bond Polypeptides are built through a series of dehydration (condensation) reactions The OH and H groups are removed from the carboxyl group of one amino acid and the amino group of the next amino acid The removal of water forms a covalent peptide bondPeptide bondsMolecular Biology大连理工大学 Polypeptides contain N termini (amino terminus) and C termini (carboxyl terminus ), usually ranging from 100-1500 aa.Peptide bondsN-terminus and C-terminusN terminusC terminusMolecular Biology大连理工大学Some of the Diverse Functions of ProteinsMolecular Biology大连理工大学Primary StructureuAmino acid sequence of a proteinuWritten from amino terminus to carboxyl terminusuCoded for by nucleotide sequence from the mRNA, therefore represents the commands of the DNA sequenceuSequence determines secondary, tertiary and quaternary structures, as well as protein-protein and protein-nucleic acid recognitionsuA single substitution may result in a protein that is not folded properly (hemoglobin)Molecular Biology大连理工大学 Secondary structureuFolding into helix, b sheet or random coiluInvolves local interactions between amino acidsuInteractions lead to the formation of the helix or b sheet己糖激酶 Molecular Biology大连理工大学 helixuHelix is derived from repeating polymers and results in 3.6 amino acids per turn, bringing every 4th amino acid in proximityuL (leu), M(met), and E(glu) are strong helix formersuG(gly) and P(pro) are helix breakers and are involved in bends and turns in helicesMolecular Biology大连理工大学The b b sheetuMaximized by hydrogen bonding, but between two polypeptides or two different segments of one polypeptideuI(ile), V(val), and F(phe) are strong sheet formersuIf the two strands linked are aligned with both amino and both carbonyl groups running in the same direction, then this is a parallel sheetuIf the two strands run in opposite directions, this is an anti-parallel sheetMolecular Biology大连理工大学Motifs Motifs: commonly occurring secondary structures b-b motif Hairpin turn motif Helix-turn-helix motifMolecular Biology大连理工大学Tertiary structureudepends more on the R groups than the amino or carboxyl groupsuHydrophobic domains tend to associate with one another and are often found in the inner sections of the proteinMolecular Biology大连理工大学Stabilization of tertiary structuresuNoncovalent bondsuHydrogen bonds between appropriate R groupsuElectrostatic interactions between charged R groupsuHydrophobic interactions between nonpolar R groupsuCovalent bondsuDisulfide bond between two cysteine residuesuDisulfide bonds are important for stabilizing both tertiary and quaternary structuresMolecular Biology大连理工大学Molecular Biology大连理工大学Protein domain Most globular(球状) proteins consists of several domains(结构域) A domain is a discrete, locally folded unit of tertiary structure 50-350 residues Each domain has a specific function within the protein, such as metal binding domain of enzymes Small globular proteins tend to have a single domain Large globular proteins have multiple domainsMolecular Biology大连理工大学Quaternary StructureLevel of protein organization related to subunit interactions and assembliesMolecular Biology大连理工大学Molecular Biology大连理工大学Proteins structuresLevel of structureBasis of StructureBonds involvedPrimaryAmino acid sequenceCovalent bondsSecondaryFolding into helix, b sheet or random coilHydrogen bondsTertiary3D folding of a single polypeptideHydrogen and disulfide bonds, electrostatic and hydrophobic interactionsQuaternaryAssociation of 2 or more folded subunitsSame as tertiaryMolecular Biology大连理工大学Nucleic AcidsMolecular Biology大连理工大学Nucleic Acids Linear polymers of nucleotides Store, carry, and aid in the transmission of genetic information Differ in type of sugar used and length of polymers Deoxyribonucleic acid (DNA) Ribonucleic acid (RNA) Sugar in each case is the pentose(五元糖)Molecular Biology大连理工大学Background In 1869, Friedrich Meischer isolated DNA from fish sperm and the pus of open wounds. Since it came from nuclei, Meischer named this new chemical, nuclein(核素）. Subsequently the name was changed to nucleic acid and lastly to deoxyribonucleic acid (DNA). In 1914 , Robert Feulgen（福尔根） discovered that fuchsin（品红） dye stained DNA. DNA was then found in the nucleus of all eukaryotic cells. Molecular Biology大连理工大学Background During the 1920s, biochemist P.A. Levene analyzed the components of the DNA molecule, which contained four nitrogenous bases: cytosine, thymine, adenine, and guanine; deoxyribose sugar; and a phosphate group. He concluded that the basic unit (nucleotide) was composed of a base attached to a sugar and that the phosphate also attached to the sugar. Unfortunately, he incorrectly concluded that the proportions of bases were equal and that there was a tetranucleotide(四核苷酸） that was the repeating structure of the molecule. Molecular Biology大连理工大学Components of Nucleic acidsEach nucleotide has the three following components A cyclic five-carbon sugar: ribose in RNA，or deoxyribose(脱氧核糖) in DNA. A purine(嘌呤) or pyrimidine（嘧啶） base attached to 1-carbon atom of sugar by N-glycosylic(糖基) bond. A phosphate attached to the 5carbon of the sugar by a phosphoester(磷酯) linkage.Molecular Biology大连理工大学Components of Nucleic acids: Phosphate and pentosePurines and pyrimidines can form chemical linkages with pentose (5-carbon) sugars. The carbon atoms on the sugars are designated 1, 2, 3, 4 and 5. It is the 1 carbon of the sugar that becomes bonded to the nitrogen atom at position N1 of a pyrimidine or N9 of a purine. DNA precursors contain the pentose deoxyribose. RNA precursors contain the pentose ribose (which contains an additional hydroxyl group(羟基 ) at the 2 position)Molecular Biology大连理工大学Nucleotides Are the Monomers That Create Polymers of DNA and RNABases are insideSugar and P are outsideOf the helixMolecular Biology大连理工大学hydrogen bonding in Nucleic Acids REQUIRED FOR SPECIFICITY OF BASE PAIRING Recognition of component molecules depends on features of purines and pyrimidines Carbonyl groups and nitrogen are capable of hydrogen bonding Hydrogen bonds form between A:T or A:U or between G:C NOT VERY IMPORTANT IN DNA STABILIZATION HYDROPHOBIC FORCES ARE THE MOST IMPORTANTMolecular Biology大连理工大学Molecular Biology大连理工大学DNA conformations DNA can have several conformations. The most common one is called B-DNA. B-DNA is a right-handed double helix with a wide and narrow groove(槽). The bases are perpendicular(垂直) to the helix axis.B-DNAMolecular Biology大连理工大学DNA conformations DNA can also be found in the A form in which the major groove is very deep and the minor groove is quite shallow. A formMolecular Biology大连理工大学DNA conformations A very unusual form of DNA is the left-handed Z-DNA. Z-DNA forms excellent crystals.Z-DNAMolecular Biology大连理工大学RNA conformation RNA can form double stranded duplexes. These duplexes are in the A conformation because the 2OH precludes the B conformation. More commonly, RNA is single stranded and can form complex and unusual shapes. A-RNAMolecular Biology大连理工大学CarbohydratesMolecular Biology大连理工大学Carbohydratesw Monosaccharides - simple sugars with multiple OH groups. Based on number of carbons (3, 4, 5, 6), a monosaccharide is a triose, tetrose, pentose or hexose.w Disaccharides - 2 monosaccharides covalently linked.w Oligosaccharides - a few monosaccharides covalently linked. w Polysaccharides - polymers consisting of chains of monosaccharide or disaccharide units. I (CH2O)n orH - C - OHIStructure: Molecular Biology大连理工大学MonosaccharidesAldoses (e.g., glucose) have an aldehyde group at one end.Ketoses keto . CCOHHCHHOCOHHCOHHCH2OHD-glucoseOH CHHOCOHHCOHHCH2OHCH2OHCOD-fructoseMolecular Biology大连理工大学D and L sugarsuFor sugars with more than one chiral center, D or L refers to the asymmetric C farthest from the aldehyde or keto group. uMost naturally occurring sugars are D isomers.D and L sugars are mirror images of one another. O H O H C C H C OH HO C H HO C H H C OH H C OH HO C H H C OH HO C H CH2OH CH2OH D-glucose L-glucose Molecular Biology大连理工大学Glycosidic BondsThe hydroxyl group of one sugar and the hydroxyl group of another sugar or some other compound can join together, splitting out water to form a glycosidic bond: R-OH + HO-R R-O-R + H2OE.g., methanol reacts with an OH on glucose to form methyl glucoside. OHHOHHOHOHOHHHOH-D-glucopyranoseOHHOHHOHOCH3OHHHOHmethyl-D-glucopyranoseCH3-OH+methanolH2OMolecular Biology大连理工大学Cellobiose, a product of cellulose breakdown, is the otherwise equivalent b anomer (O on C1 points up). The b(14) glycosidic linkage is represented as a zigzag, but one glucose is actually flipped over relative to the other. HOOHHOHHOHCH2OHHOHOHHOHHOHCH2OHHOHH123546123456maltoseHOOHHOHHOHCH2OHHOOHHHOHHOHCH2OHHHHO123456123456cellobioseDisaccharides:Maltose, a cleavage product of starch, is a disaccharide with an (14) glycosidic link between the C1 OH & C4 OH of 2 glucoses. It is the anomer, because the O on C1 points down.Molecular Biology大连理工大学DisaccharidesOther common disaccharides include: Sucrose, common table sugar, has a glycosidic bond linking the anomeric hydroxyls of glucose & fructose. Because the configuration at the anomeric C of glucose is (O points down from ring), the linkage is (12). The full name of sucrose is -D-glucopyranosyl-(12)-b-D-fructopyranose.) Lactose, milk sugar, is composed of galactose & glucose, with b(14) linkage from the anomeric OH of galactose. Its full name is b-D-galactopyranosyl- (14)-D-glucopyranoseMolecular Biology大连理工大学Polysaccharides 多糖多糖uThey are very complex molecules because sometimes covalent bonds 共价键 occur between many parts of carbon atoms, allowing one sugar unit to be joined to more than two other sugars, which results in the formation of highly branched macromolecules.uPolysacch