00Note Set 4 1 NUCLEIC ACIDS The Nature of Nucleic Acids What is the role nucleic acids play in the formation of proteins and cellular structure? THE TWO TYPES OF NUCLEIC ACID: DNA AND RNA (see Fig 4.1) phosphate can attach to 3' and 5' hydroxyl to form phosphodiester link phosphate is a strong acid, pka 1 ~1 (Table 4.1) nucleotides are composed of a nitrogenous base linked to a suger (at 1' carbon), with at least one phosphate group attached to the sugar 1. (Fig 4.2) Purine bases: adenine A and guanine G Purine nucleosides: adenosine A and guanosine G Purine nucleotides: adenylic acid/adenosine monophosphate AMP and guanylic acid/guanosine monophosphate GMP Pyrimidine bases: cytosine C and (urasil U or thymine T) Pyrimidine nucleosides: cytidine C and (uridine U or deoxythymidine T) Pyrimidine nuclotides: cytidylic acid/cytidine monphosphateand uridylic acid/uridine monophosphate UMP or deoxythymidylic acid/deoxythymidine monophosphate TMP 2. Primed numbers refer to the sugar (ribose), and unprimed numbers refer to the nitrogenous bases 3. Phosphate group may be attached to eithe the 3 or 5 C of the ribose 4. Free nuclotides are anions, and a usually associated with Mg 2+ 5. Bases can tautomerize, form tautomerc forms = structural isomers with H atoms and double bonds in different locations (Fig 4.4) 6.(Fig 4.5) UV spectra of ribons, usually look at 260-280 for RNA and DNA Stability and formation of phosphodiester bond Could be a simple dehydration, but G +25 kj/mol (Fig 4.1, 4.3), so nucleoside triphosphates are used ( G = -31kJ/mol). Overall, -6 kj/mol (Fig 4.6,4.7) Hydrolysis is favorable, but extremely slow so DNA is stable
00Note Set 4 2 RNA much more susceptible to hydrolysis especially if alkaline, because of 2' OH Acid catalyzes hydrolysis of DNA and RNA, keep away from very low ph. Nucleases catalyze hydrolysis of peptide bonds in RNA and DNA, such as restriction enzymes cutting specific bonds in DNA NUCLEIC ACID STRUCTURE: Primary Structure 1. Phosphates are acidic and at physiological ph are negatively charged 2. Phosphate is esterified to two ribose units by a phosphodiester bond 3. Each nuclotide incorporated is called a nucleotide residue 4. There is a 5 end (left) and a 3 (right) end Note: Review Fig 4.8 and section on DNA as the genetic substance:early evidence Timeline 1866 Mendel s theory of inheritance factors 1869 Miescher isolates and characterizes nucleic acids Monotonously repeating nucleotide sequences 1944 Avery, MacLeod and McCarty showed that DNA not protein carries genetic information 1940s Beadle and Tatum proposed one gene-one enzyme theory 1958 Crick formulated central dogma of molecular biology The RNA World 1. The ribosome is a big enzyme complex The component that catalyzes the formation of a peptide bond is rrna 2. Thus, RNA can catalyze chemical reactions in addition to its function as an information carrier 3. First life was probably a primitive vesicle containing RNA Secondary and teriary structure
00Note Set 4 3 The Double Helix 1. Nucleic acid bases in keto form (tautomers are easily converted isomers that differ only in H position and double bond position) 2. Structure solved by Watson and Crick Rosalind Franklin, Linus Pauling, Jerry Donahue using x-ray diffraction data (see Tools 4A) 3. Stuctural features: Helix is right-handed, but can also be left-handed (Z DNA) Phosphates on outside/periphery to minimize charge repulsion Surface contains major and minor grooves (Fig 4.11) Two strands are antiparrallel Bases H-bonded to form planar base pair, called complementary base pairing In double stranded DNA A = T and G = C, Chargaff's rule Self-replication (Fig 4.12, and Meselson-Stahl determination of semiconservative replication 4.13, 4.14) B form predominates (see Table 4.3), more common in aqueous milieu, A form more dehydrated A and B both rotate to right, differ in pitch of helix (# of residues/turn x rise per residue) (Fig 4.15) In vivo: some viruses very small like a few thousand base pairs (there are also single stranded DNA viruses and RNA viruses) Circular DNA and supercoiling (Fig 4.18) Catalyzed by topoisomerases, sometimes called DNA gyrases Also see trna structure Fig 4.20 Review Molcular Biology section (movies in class, and Figs 4.21,4.22,4.23) The genome Transcription Translation
00Note Set 4 4 Plasticity of secondary and teriary DNA structure There are a number of different structures, we will skip most of this section. Skip: gory details of supercoiling Hairpins and cruciforms (requires palindromes, see Figs 4.27,4.28) Also Triple Helices (Fig 4.29) H-DNA: all of one strand is puines, other is all pyrimidines (Fig 4.30) Stability of 2 and 3 structure Nucleic acid denaturation (Fig 4.31) G = H - T S Favored by: 1. > S 2. Electrostatic repulsion of phosphates negative charges ( Hel < 0) BUT: G for unfolding must be positive 3. Overall positive H because of energy required to break many H-bonds and vander Waals interactions 4. T = temperature must increase 5. Can be followed by UV absorption: polymerized and packed causes lower absorption = hypochromism; when structure lost, absorption increases (4.31c) 6. Transition or melting is very sharp due to cooperative interactions, that cause cooperative transitions (Fig 4.31d) 7. High GC content causes higher T m : 3 H-bonds to break/pair (Fig 4.32) Skip superhelical energy section, just remember that it takes E to make them, a la rubber band, and E is released when SCs are unwound Read Tools of Biochemistry: X-ray diffraction (4A) and Synthesis of oligonucleotides (4B) RFLPs 1. Human DNA differs in sequence by about 1 bp every 200-500 bp
00Note Set 4 5 2. Sometimes causes loss of gain of RE site 3. Can be used to diagnose genetic disease and identify culprits (better use if for non-culprit identification) Sequences, Mutation, and Evolution 1. Can group organisms by DNA sequence relatedness 2. DNA sequence relationships (and genome structure) led to the regrouping of prokaryotes to include eubacteria and archea 3. Sudden genetic changes seen in fossil record Punc Eq = Puctuated Equilibrium (S J Gould) Genome Shock Barbara McClintock 4. Humans and chimps 98-99% identical at DNA level RECOMBINANT DNA TECHNOLOGY Genomic Libraries and Cloning 1. DNA is usually partially digested with a RE that has a 4-base recognition sequence 2. In order to cover the genome the following formula can be used to determine how many clones (colonies, plaques) the library will need to contain P = 1 - (1-f) N and to solve for N: N = log(1-p) / log(1-f) P is the probability that a fraction f in bp will contain the clone you want if the library you make has N clones (colonies, plaques) It is desirable that P =.99 Movies in class: PCR (p922) Site-Directed Mutagenesis (site-specific mutagenesis p978)