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Nucleic acids, Gene expression and Recombinant DNA technology • Describe chemical structure of DNAs • derive the concept that DNA is the carrier of genetic information • general concepts of how genes are expressed • general concept of how DNA is replicated • gene manipulation and expression The chemical components in DNA (1) Nucleotides and nucleic acids The importance of nucleotides (a) Monomeric unit of DNAsÆthe storage and expression of genetic information (b) Nucleoside triphosphate (ATP) as energy source. (c) ATP and ADT regulate the activities of numerous metabolic processes. (d) Nucleotide derivatives, nicotinamide adenine dinucleotide, flavin adenine dinucleotide, and coenzyme A, are required in many enzymatice reactions. (e) Nucleotides themselves have catalytic activities, such as ribozymes. (2) Nucleotides, nucleosides, and bases Nucleotides=phosphate + five-carbon sugar (+ nitrogenous base) Ribonucleotide: The monomeric units of RNA Deoxyribonucleotides: The monomeric units of DNA C5’ of pentose-the phosphate- C3’ of phosphate Nucleoside-absence of phosphate group NDPs and NTPs are Polyprotic Acids The D-ribose is is an important sugar used in genetic materials. It is not used as energy source but is a part of the backbone of RNA and DNA. The D-ribose undergoes cyclization, forming a five-membered ring. (1) The base linked to to C1’ of the ribose through glycosidic bond. The linkage of the base is on the same side of the ribose as does the phosphate group (called E configuration) (2) Protons from the phosphoric acid groups are dissociable; Resulting phosphate anions form tight complexes with Mg++ and Ca ++ The nitrogeous bases Planar, aromatic and heterocyclic compounds originally derived from purine or pyrimidine. Pyrimidines connect to ribose at N1 postions; Purines connect to ribose at N9 positions. The other important feature of the base is the Keto-enol tautomeric shifts Strong UV absorbance (max. 260 nm) for all bases except for cytosine. Tautomerism refers to an equilibrium between two different structure of the same compound. Usually the tautomers differ in the point of attachment of a hydrogen atom. One of the most common examples of a tautomeric system is the equilibrium between a ketone and its enol form, Structure and Nomenclature of Nucleic Acids (1) Nucleic acids are polynucleotides; (2) Nucleotides are linked by phosphodiester bridges from 3’ to 5’; (3) Polymers of ribonucleotides are ribonucleic acids, or RNA; (4) Polymers of deoxyribonucleotides are deoxyribonucleic acids, or DNA; (1) DNA are found in different cells and viruses. They code for genetic information; (2) DNA nearly always forms double helix; (3) The two strands of DNA are complementary through base-pairing interactions; (4) Chargaff’s rules: G=C and A=T G and C and A and T are complementary bases DNA base composition varied from 25% to 70% among different organisms. Double helical DNA structure The DNA structure was determined by Watson and Crick in 1953. This finding of the helical structure of the DNA also ties together the results of several diverse studies. (1) Chargaff’s rule: A=T, G=C (2) The bases are dominantly in keto tautomeric forms. (3) DNA is in helical structure. The planar aromatic bases forms a parallel rings which is parallel to the helical axis. The Watson-Crick B-DNA structure B-DNA is regarded as the biologically functional native form of DNA. The features of B-DNA are (1) Two polynucleotides strands wind about a common axis. (2) The helix is in right-handed twist and the diameter is ~20Å. (3) The bases are in the core of the helix and sugar-phosphate backbones are coiled about the helix’s periphery. (4) The two DNA strands are antiparallel. Double helical DNA structure (5) The planes of the bases are nearly perpendicular to the helix axis. (6) The complementary bases pairing of the bases result in the hydrogen bonding interactions which associate the two DNA strands in the helix. (7) The B-DNA has 10 base pairs per turn, the rise is 3.4 Å, and the pitch is 34 Å. (8) Unique Watson-Crick base pairs-A.T or C.G. These base pairs are interchangeable without disturbing helical structure. (9) The helical structure has two exterior grooves, major and minor grooves, which show the asymmetric properties of the bases. DNAÅÆ heredity (1) Transforming principle is DNA In 1928, Frederick Griffith٬Ҕషӝޑςύޑک S(virulent)ݹޤԝکR(ߚठੰࢲݹޤ)܄ݙ ΕჴᡍႵǴวεӭኧޑჴᡍႵԝǴԶว ࢲS form(virulent)ݹޤ. In 1994, Oswald Avery, Colin Macleod, and Maclyn McCartyวtransforming principleࣁ DNAǶ (2) Bacteriophage T2གࢉჴᡍ In 1952, Alfred Hershy and Martha Chase٬Ҕࢲ ᡏ32PǴ32S radio labeled T2ᏘᡏǴགࢉεဉఎ ࡕǴϩࡕжᏘᡏܫ܄ނۓ፦ࣁ30% 32P, 1% 32S, ຓჴѝԖᏘᡏDNAࣁᒪࡕж܌ ሡނޑ፦Ƕ Denaturation and renaturation of DNA When solution of DNA is heated, the native DNA structure collapsed. (1) Each strand is in flexible and fluctuating conformation state known as random coil. (2) Physical properties change, such as viscosity, and UV absorbance. Hyperchromic effect: The UV absorbance of the denatured DNA increases by 40% in all wavelength, whereas the absorbance curve does not change. ѓკຓჴDNA denaturation is a cooperative processӢࣁ֎Ӏ܄፦ׯᡂჹᔈࡋྕޑጄൎࡐઞ. ஒRenatured DNAჴᡍచҹှନࡕ, DNAӣᙟ ᚈިconformation.ӕ, RNA-DNA hybridization Ψёаԋᚈި่ᄬǶ Gene expression and replication The central dogma of Molecular Biology DNAÆ transcription (m-RNA) Æ (modification) Æ translation (protein) Other routes are possible RNAÆ RNA RNAÆ DNA (reverse transcription) DNAÆ protein (?) ՠࢂproteinÆXRNA or DNA Transcription: Synthesis of RNA The enzyme, RNA polymerase, synthesizes RNA in the 5’Æ 3’ direction and appends NTPs (ATP, GTP, CTP, UTP) to the free 3’OH group of the grouping RNA. The process of transcriptional regulation The characteristics of the sequence of the DNA template (1) Initiation site (2) Control sites: regulates transcription on or off by proteins known in prokaryotes as activator or repressor and in eukaryotes as transcription factor. (3) termination E. Coli lac operon genes transcriptional control Translation: synthesis of protein Proteins are synthesized under the direction of the corresponding mRNA. The ribosome machinery for making proteins comprises two-thirds RNA and on-third protein (~2500 kD in prokaryotes and ~4200 kD in eukaryotes) mRNAύޑΟঁׇӈᆀϐࣁcodon(ӵፐҁύ)߄ޑǴჹ ᔈډനಖᙯࡕ܌ᇙԋޑữ୷ለǶӧ3‘ᆄሒௗჹᔈữ୷ለ Aminoacyl-tRNAԿribosomeՏǴҗܭtRNAڀԖanticodonׇӈǴӢԜပԿribosomeޑA-siteǶribosomeϯ ϸᔈஒ peptidyl-t-RNA౽Կ Aminoacyl-tRNAǴԶҔၸޑ tRNAҗP-site ౽рǶ Genetic codes Ӣࣁ3ঁDNAׇӈჹᔈঁữ୷ለǴ܌ аᕴӅԖ64 ঁcodonsǴځύ61ঁჹᔈ ډữ୷ለǴԶSTOP codon ࣁUAA, UAG, ϷUGAǶMet ǴTrpѝԖঁჹ ᔈcodon ;Leu, Ser, ArgԖϤঁჹcodon, ӢԜgenetic codeᆀϐࣁdegenerateǴՠ ࢂჹᔈӕঁữ୷ለޑcodonৡ౦ӧಃ ΟঁਡለǶ The first synthesized a.a. is Methionine Even though the initiation codon is AUG, the tRNA that recognizes this initiation codon is different from the tRNA that delivers a polypeptide’s internal Met residue to the ribosome. How did the ribosome select the initiation codon from among the many AUGs? (1) In prokaryote, ӧinitiation codon 5’ᆄෞׇӈᡣribosomeᒣᇡଆۈcodonǶ (2) In eukaryote, ӧmRNAޑ5‘ᆄcapଆࡕۈಃঁAUGջࣁଆۈ. ߕຏ: tRNAҗamino-acyl-tRNA synthetasesஒa.a.к༤ԿtRNAǶ DNA replication DNA replication requires (1) Deoxynucleoside triphosphate (dNTP) (2) DNA polymerase: can only extends(5’Æ3’) an existing polynucleotide (primer) that is base paired to the DNA’s template strand. (3) Primers are RNA In E. Coli, these primers are made by both RNA polymerase and primase. (4) Both of the DAN strands are simultaneously replicated at the replication fork where (i) Leading strand is synthesized in 5’Æ3’ direction. (ii) The complementary strand (the lagging strand) is synthesized discontinuously. DNA replication DNA polymeraseIII in E. Coli is a DNA replicase and synthesizes the leading strand and most of the lagging DNA strand. DNA polymerase I in E. Coli has both the DNA polymerase activity and 5’Æ3’exonucleonase activity. Pol I’s 5’Æ3’ exonuclease and DNA polymerase activity work in concert. It removes the RNA primers and replacing them with DNA. The synthesis of the lagging strand is completed by sealing the nicks between multiple discontinuously synthesized segments. DNA ligase covalently linked adjacent 3’-OH abd 5’-phosphate group and sealed the gap. DNA is semi-conservatively replicated The semiconservative nature of DNA replication was demonstrated in 1958 by Mathew Meselson. The DNA replication results in two molecules of duplex DNA, each consisting of one strand from the parent molecule and a newly synthesized complementary strand. Experiment: (1) To grow E. Coli using 15NH4CL as a sole nitrogen source for 14 generations. (2) Transfer cell to 14N-containing medium. (3) Equilibrium density gradient ultracentrifugation method to monitor DNA as a function as cell growth. Error correction in DNA replication In E.Coli RNA polymerase error: 1/104 DNA polymerase error: 1/108~1010 In DNA replication, the error was guarded by (1) Using RNA primers to increase the DAN replication fidelity of the lagging strand. (2) Both pol I and pol III have 3’Æ5’ exonuclease activities, which degrade the newly synthesized 3’-end of a daughter strand. This reaction is activated by non-Watson-Crick base pairing. (3) A series of enzymes were used to detect and correct residual erros in replication and DNA damages caused by UV radiation, mutagens, and spotaneously hydrolysis. Then, in E. Coli, Pol I replaces the damage DNA segments excised by these enzymes. The polymerase chain reaction In 1985, Kerry Mullis devised the PCR method, a basis of “cell-free molecular cloning”. The applications of PCR The principle of PCR A heated denatured DNA sample is incubated with heat stable DNA polymerase, dNTP, and two oligonucleotide primers. The primer sequences flanked the DNA sequence of interest to direct the DNA polymerase to synthesize new complementary strands. Multiple cycles of heat denaturation at 95 ºC and elongation at lower temperature amplify the gene of interest to multiple copies. *** beware of the sample contamination Applications: (1) Diagnosis of infectious diseases and gene mutations (2) Amplify RNA RNAÆ reverse transcription to generate DNA ÆPCR (3) Asymmetric PCR Single strand DNA PCR with one primer (4) Nested PCR PCR with one pair of primersÆ product goes through second round of PCR with a second pair of primers annealed to the target fene within its ampified region. (5) Direct sequencing Restriction endonuclease To use restriction endonucleases to manipulate genes with precisely defined sequence. Type I, III r.e.: have both the endonuclease and the methylase activity. Type I r.e. cleaves DNA at a possibly 1000 bp from the recognition site. Type III cleaves 24 to 26 bp distant from the recognition site. Type II r.e.: cleave DNAs at specific sites within the recognition sequence. The resulting cut DNA sequence may be with cohesive or sticky ends or blunt ends. The recognition sequence may well be in palindromic sequence. Cloning vectors (1)Plasmid-based vectors Plasmids are circular DNA about ~5000 bp in size. • Plasmid copy number may be subjected to stringent control of one to few copies per cell, or to relaxed control of 10 to 700 copies per cell. • Antibiotic selection • Polylinker site for gene insertion (2) Virus based vectors Bacteriophages O vector is used to clone DNAs of up to 16 kb. The gene of interest is inserted into the central third of the 48.5-kb genome. Filamentous bacteriophage M13 baculovirus (3) YAC and BAC vectors Yeast artificial chromosomes (YACs) and Bacterial artificial chromosomes (BACs) vectors were used to amplified long DNA segments. The former are linear DNA segments required for replication in yeast, whereas the latter is used in E. Coli. Gene manipulation Southern blot: identify seqcific DNA sequence • Gel electrophoresis of double stranded DNA sample • the gel is soaked in 0.5 M NaOH to convert the DNA to the single-stranded form. • blot on nitrocellulase membrane • soaked in solution containing 32P-label single stranded DNA or RNA probe • autoradiogram Construct of the genomic library Shotgun cloning to construct genomic library: Chromosom DNA is cleaved to fragments of clonable size, and inserted in cloning vector to construct plasmid-, phage-, or cosmid-based genmic libraries. Selection of the correct clones was done by in-situ hybridization. The correct clones were subject to a process called “chromosome walking” to complete the DNA sequencing. 123 RNA molecules RNA occurs in multiple forms (can be double helix but not necessary) and copies; Messenger RNA codes template for protein synthesis; Ribosomal RNA constitute the catalytic core of the ribosome; Transfer RNA is the adaptor between nucleic acids and proteins; Small nuclear RNA are essential component of splicesome; microRNA regulates gene expression. RNA but not DNA is susceptible to base-catalyzed hydrolysis