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Chem 4311 Chapter10 12 Nucleic Acid 10-9-12
Chem 4311 Chapter10 12 Nucleic Acid 10-9-12
Chem 4311 Chapter10 12 Nucleic Acid 10-9-12
We have discovered the secret of life. Francis Crick, to patrons of The Eagle, a pub in Cambridge, England (1953)
Francis Crick (right) and James Watson (left) point out features of their model for the structure of DNA.
Know the basic structures Pyrimidines Cytosine (DNA, RNA) Uracil (RNA) Thymine (DNA) Purines Adenine (DNA, RNA) Guanine (DNA, RNA)
Figure 10.3 The common pyrimidine bases cytosine, uracil, and thymine in the tautomeric forms predominant at pH 7.
Figure 10.4 The common purine bases adenine and guanine in the tautomeric forms predominant at pH 7.
The Properties of Pyrimidines and Purines Can Be Traced to Their Electron-Rich Nature
The aromaticity and electron-rich nature of pyrimidines and purines enable them to undergo keto-enol tautomerism The keto tautomers of uracil, thymine, and guanine predominate at pH 7 By contrast, the enol form of cytosine predominates at pH 7 Protonation states of the nitrogens determines whether they can serve as H-bond donors or acceptors Aromaticity also accounts for strong absorption of UV light
The Properties of Pyrimidines and Purines Can Be Traced to Their Electron-Rich Nature
The Properties of Pyrimidines and Purines Can Be Traced to Their Electron-Rich Nature
The base is linked to the sugar via a glycosidic bond The carbon of the glycosidic bond is anomeric Named by adding -idine to the root name of a pyrimidine or -osine to the root name of a purine Conformation can be syn or anti Sugars make nucleosides more water-soluble than free bases
Figure 10.11 Structures of the four common ribonucleotides AMP, GMP, CMP, and UMP. Also shown: 3-AMP.
Figure 10.13 Formation of ADP and ATP by the succesive addition of phosphate groups via phosphoric anhydride linkages. Note that the reaction is a dehydration synthesis.
Figure 10.13 Formation of ADP and ATP by the succesive addition of phosphate groups via phosphoric anhydride linkages. Note that the reaction is a dehydration synthesis.
Nucleoside 5'-triphosphates are indispensable agents in metabolism because their phosphoric anhydride bonds are a source of chemical energy Bases serve as recognition units ATP is central to energy metabolism GTP drives protein synthesis CTP drives lipid synthesis UTP drives carbohydrate metabolism
Figure 10.14 Phosphoryl, pyrophosphoryl, and nucleotidyl group transfer, the major biochemical reactions of nucleotides. Phosphoryl group transfer is shown here.
Figure 10.14 Phosphoryl, pyrophosphoryl, and nucleotidyl group transfer, the major biochemical reactions of nucleotides. Pyrophosphoryl group transfer is shown here.
Figure 10.14 Phosphoryl, pyrophosphoryl, and nucleotidyl group transfer, the major biochemical reactions of nucleotides. Nucleotidyl group transfer is shown here.
Figure 10.15 3',5'Phosphodiester bridges link nucleotides together to form polynucleotide chains. The 5'ends of the chains are at the top; the 3'-ends are at the bottom. RNA is shown here.
Figure 10.15 3,5phosphodiester bridges link nucleotides together to form polynucleotide chains. The 5-ends of the chains are at the top; the 3-ends are at the bottom. DNA is shown here.
DNA - one type, one purpose RNA - 3 (or 4) types, 3 (or 4) purposes ribosomal RNA - the basis of structure and function of ribosomes messenger RNA - carries the message for protein synthesis transfer RNA - carries the amino acids for protein synthesis Others:
Small nuclear RNA Small non-coding RNAs
Figure 10.16 The antiparallel nature of the DNA double helix. The two chains have opposite orientations.
Figure 10.17 The Watson-Crick base pairs A:T and G:C. A:T is shown here.
Figure 10.17 The Watson-Crick base pairs A:T and G:C. G:C is shown here.
Figure 10.18 Replication of DNA gives identical progeny molecules because base pairing is the mechanism that determines the nucleotide sequence of each newly synthesized strand.
Transcription product of DNA In prokaryotes, a single mRNA contains the information for synthesis of many proteins In eukaryotes, a single mRNA codes for just one protein, but structure is composed of introns and exons
Figure 10.20 Transcription and translation of mRNA molecules in prokaryotic versus eukaryotic cells. In prokaryotes, a single mRNA molecule may contain the information for the synthesis of several polypeptide chains within its nucleotide sequence.
Figure 10.20 Transcription and translation of mRNA molecules in prokaryotic versus eukaryotic cells. Eukaryotic mRNAs encode only one polypeptide but are more complex.
Eukaryotic mRNA
DNA is transcribed to produce heterogeneous nuclear RNA (hnRNA) mixed introns and exons with poly A intron = intervening sequence exon = coding sequence poly A tail - stability? Splicing produces final mRNA without introns
Ribosomal RNA Provides the Structural and Functional Foundation for Ribosomes
Ribosomes are about 2/3 RNA, 1/3 protein rRNA serves as a scaffold for ribosomal proteins The different species of rRNA are referred to according to their sedimentation coefficients rRNAs typically contain certain modified nucleotides, including pseudouridine and ribothymidylic acid The role of ribosomes in biosynthesis of proteins is treated in detail in Chapter 30 Briefly: the genetic information in the nucleotide sequence of mRNA is translated into the amino acid sequence of a polypeptide chain by ribosomes
Ribosomal RNA Provides the Structural and Functional Foundation for Ribosomes
Transfer RNAs Carry Amino Acids to Ribosomes for Use in Protein Synthesis
Small polynucleotide chains - 73 to 94 residues each Several bases usually methylated Each a.a. has at least one unique tRNA which carries the a.a. to the ribosome 3'-terminal sequence is always CCA-3-OH. The a.a. is attached in ester linkage to this 3-OH. Aminoacyl tRNA molecules are the substrates of protein synthesis
Glycolaldehyde has been detected at the center of the Milky Way and could be a precursor of ribose and glucose.
The Chemical Differences Between DNA and RNA Have Biological Significance
The Chemical Differences Between DNA and RNA Have Biological Significance
Why does DNA contain thymine? Cytosine spontaneously deaminates to form uracil Repair enzymes recognize these "mutations" and replace these Us with Cs But how would the repair enzymes distinguish natural U from mutant U? Nature solves this dilemma by using thymine (5methyl-U) in place of uracil
Figure 10.27 Alkaline hydrolysis of RNA. Nucleophilic attach by OH- on the P atom leads to 5'-phosphoester cleavage.
Figure 10.27 Alkaline hydrolysis of RNA. Nucleophilic attack by OH- on the P atom leads to 5'-phosphoester cleavage. Random hydrolysis of the cyclic phosphodiester intermediate gives a mixture of 2'- and 3'-nucleoside monophosphate products.
Figure 10.28 Cleavage in polynucleotide chains. Cleavage on the a side leaves the phosphate attached to the 5'position of the adjacent nucleotide. b-side hydrolysis yields 3'-phosphate products.
Restriction Enzymes
Bacteria have learned to "restrict" the possibility of attack from foreign DNA by means of "restriction enzymes" Type II and III restriction enzymes cleave DNA chains at selected sites Enzymes may recognize 4, 6 or more bases in selecting sites for cleavage An enzyme that recognizes a 6-base sequence is a "six-cutter"
EcoRI -----------GAATTC--------------------CAATTG----------
BamHI -----------GGATCC--------------------CCTAGG----------
MstI
-----------TGCGCA--------------------ACGCGT----------
11.1 How Do Scientists Determine the Primary Structure of Nucleic Acids? Two simple tools have made nucleic acid sequencing easier than polypeptide sequencing:
The type II restriction endonucleases that cleave DNA at specific oligonucleotide sites Gel electrophoresis, which is capable of separating nucleic acid fragments that differ from one another in length by just a single nucleotide
Figure 11.1 DNA replication yields two daughter DNA duplexes identical to the parental DNA molecule.
Figure 11.2 Primed synthesis of a DNA template by DNA polymerase, using the four deoxynucleoside triphosphates as the substrates.
Emerging Technologies to Sequence DNA are Based on Single-Molecule Sequencing Strategies Growing demand for sequence information is driving the development of faster and cheaper methods of DNA sequencing One technique involves passing a single strand of DNA through a graphene monolayer pore, measuring the change in electrical conductance (ion flow) through the pore Each base alters electrical conductance in a subtle but different way, facilitating the reading of sequence
11.2 What Sorts of Secondary Structures Can DoubleStranded DNA Molecules Adopt? Polynucleotide strands are flexible Each deoxyribose-phosphate segment of the backbone has six degrees of freedom (Fig 11.4a) Furanose rings are not planar but instead adopt puckered conformations, four of which are shown in Figure 11.4b A seventh degree of freedom per nucleotide arises because of free rotation about the C1'-N glycosidic bond This freedom allows the plane of the base to rotate relative to the path of the polynucleotide strand
11.2 What Sorts of Secondary Structures Can DoubleStranded DNA Molecules Adopt?
Figure 11.6 The six degrees of freedom in the deoxyribose-PO4 units of the polynucleotide chain.
11.2 What Sorts of Secondary Structures Can DoubleStranded DNA Molecules Adopt?
Figure 11.7
(a) Double-stranded DNA as an imaginary ladderlike structure. (b) A simple right-handed twist converts the ladder to a helix.
11.2 What Sorts of Secondary Structures Can Double-Stranded DNA Molecules Adopt?
The stability of the DNA double helix is due to: Hydrogen bonds between base pairs Electrostatic interactions mutual repulsion of phosphate groups, which makes them most stable on the helix exterior Base-pair stacking interactions Right-twist closes the gaps between base pairs to 3.4 A (0.34 nm) in B-DNA
Comparison of A, B, Z DNA
See Table 11.1 A: right-handed, short and broad, 2.3 , 11 bp per turn B: right-handed, longer, thinner, 3.32 , 10 bp per turn Z: left-handed, longest, thinnest, 3.8 , 12 bp per turn See Figure 11.11
Intercalating Agents Distort the Double Helix The double helix is a very dynamic structure Because it is flexible, aromatic macrocycles flat hydrophobic molecules composed of fused, heterocyclic rings, can slip between the stacked pairs of bases The bases are force apart to accommodate these intercalating agents
Ethidium bromide Acridine orange Actinomycin D
Single-Stranded DNA Can Renature to Form DNA Duplexes Denatured DNA will renature to re-form the duplex structure if the denaturing conditions are removed Renaturation requires reassociation of the DNA strands into a double helix, a process termed reannealing For this to occur, the strands must realign so that their complementary bases are once again in register and the helix can be zippered up
Nucleic Acid Hybridization: Different DNA Strands of Similar Sequence Can Form Hybrid Duplexes
If DNA from two different species are mixed, denatured, and allowed to cool slowly, hybrid duplexes may form, provided the DNA from one species is similar in sequence to the other The degree of hybridization is a measure of the sequence similarity between the two species 25% of the DNA from a human forms hybrids with mouse DNA, implying some sequence similarity Hybridization is a common procedure in molecular biology for identifying specific genes and for revealing evolutionary relationships
Nucleic Acid Hybridization: Different DNA Strands of Similar Sequence Can Form Hybrid Duplexes
Figure 11.22 Solutions of human DNA (red) and mouse DNA (blue) are mixed and denatured; then, the single strands are allowed to reanneal. About 25% of human DNA forms hybrid duplexes with mouse DNA.
Supercoiled DNA is characterized by a Linking Number (L), Twist (T), and Writhe (W)
Figure 11.24 Linking number (L) is sum of twist (T) and writhe (W)
Supercoiled DNA is characterized by a Linking Number (L), Twist (T), and Writhe (W)
Figure 11.24 Linking number (L) is sum of twist (T) and writhe (W)
Negative supercoils cause a torsional stress on the molecule, so the molecule tends to unwind. Negative supercoiling makes it easier to separate DNA strands and access the information encoded by the sequence.
Figure 11.25 A model for the action of bacterial DNA gyrase (topoisomerase II).
Figure 11.25 Conformational changes in the enzyme allow an intact region of the DNA duplex to pass between the cut ends. The cut ends are religated (3), and the covalently complete DNA duplex is released from the enzyme. The circular DNA now contains two negative supercoils (4).
Pairs of histones H2A, H2B, H3, and H4 aggregate to form octameric core structures; the DNA helix is wound around these core octamers, creating nucleosomes.
Nucleosome Structure
Chromatin, the nucleoprotein complex, consists of histones and nonhistone chromosomal proteins Histone octamer structure has been solved (without DNA by Moudrianakis, and with DNA by Richmond) Nonhistone proteins are regulators of gene expression
Figure 11.30 A model for chromosome structure, human chromsome 4, showing nucleosomes in the beads on a string motif.
Figure 11.30 A model for chromosome structure, human chromsome 4. The 30-nm fiber is created when the array of nucleosomes adopts a two-start helical conformation.
Figure 11.30 A model for chromosome structure, human chromsome 4. The 30 nm filament forms long DNA loops of variable length, each containing on average between 60,000 and 150,000 bp.
Figure 11.30 A model for chromosome structure, human chromsome 4. Electron microscopic analysis of chromosome 4 suggests that 18 loops are arranged radially about the circumference of a single turn to form a miniband unit of the chromosome.
Figure 11.30 A model for chromosome structure, human chromsome 4. Approximately a million minibands are arranged along a central axis in each of the chromatids of chromosome 4 that form at mitosis.
Figure 11.31 SMC protein architecture and function. SMC proteins range from 115 to 165 kD in size.
Ribosomal RNA
Ribosomes synthesize proteins All ribosomes contain large and small subunits rRNA molecules make up about 2/3 of ribosome High intrastrand sequence complementarity leads to extensive base-pairing Secondary structure features seem to be conserved, whereas sequence is not There must be common designs and functions that must be conserved
12.1 What Does It Mean To Clone? Clone: a collection of molecules or cells, all identical to an original molecule or cell
To "clone a gene" is to make many copies of it for example, in a population of bacteria Gene can be an exact copy of a natural gene Gene can be an altered version of a natural gene Recombinant DNA technology makes it possible
Plasmids Are Very Useful in Cloning Genes Plamids are naturally-occurring extrachromosomal DNA Plasmids are circular dsDNA Plasmids can be cleaved by restriction enzymes, leaving sticky ends Artificial plasmids can be constructed by linking new DNA fragments to the sticky ends of plasmid These recombinant molecules can be autonomously replicated, and hence propagated
Cloning Vectors
Cloning vectors are plasmids that can be modified to carry new genes Plasmids useful as cloning vectors must have a replicator (origin of replication) a selectable marker (antibiotic resistance gene) a cloning site (site where insertion of foreign DNA will not disrupt replication or inactivate essential markers)
Chimeric Plasmids
Named for mythological beasts with body parts from several creatures
After cleavage of a plasmid with a restriction enzyme, a foreign DNA fragment can be inserted Ends of the plasmid/fragment are joined to form a "recombinant plasmid" Recombinant plasmid can replicate when placed in a suitable bacterial host
Figure 12.3 The use of linkers to create tailor-made ends on cloning fragments.
Directional Cloning
Often one desires to insert foreign DNA in a particular orientation This can be done by cleaving the plasmid with two different restriction enzymes Cleave the foreign DNA with same two restriction enzymes Foreign DNA can only be inserted in one direction
Directional Cloning
Figure 12.4 Directional cloning. DNA molecules whose ends have different overhangs can be used to form chimeric constructs in which the foreign DNA can enter the plasmid in only one orientation.
Figure 12.5 A typical bacterial transformation experiment. Here pBR322 is the cloning vector.
Shuttle Vectors Are Plasmids That Can Propagate in Two Different Organisms
Shuttle vectors are plasmids capable of propagating and transferring (shuttling) genes between two different organisms.
Figure 12.6 A typical shuttle vector. LEU2+ is a gene in the yeast pathway for leucine biosynthesis.
A DNA library is a set of cloned DNA fragments that together represent the genes of a particular organism
Any particular gene may represent a tiny, tiny fraction of the DNA in a given cell Can't isolate it directly Trick is to find the fragment or fragments in the library that contain the desired gene
Colony Hybridization
A way to screen plasmid-based genome libraries for a DNA fragment of interest Host bacteria containing a plasmid-based library of DNA fragments are plated on a petri dish and allowed to grow overnight to form colonies Replica of colonies on the dish made with a nitrocellulose disc Disc is treated with base or heated to convert dsDNA to ssDNA and incubated with a labeled probe Colonies that bind probe (labeled with 32P or other tag) hold the fragment of interest
The Southern blotting technique involves the transfer of electrophoretically separated DNA fragments to an absorbent sheet and subsequent detection of the specific DNA sequences.
Figure 12.10 Reverse transcriptase-driven synthesis of cDNA from oligo(dT) primers annealed to the poly(A) tails of purified eukaryotic mRNA.
Figure 12.11 Gene chips (DNA microarrays) in the analysis of gene expression.
Figure 12.12 Expression vectors carrying the promoter recognized by the RNA polymerase of bacteriophage SP6 are useful for the production of multiple RNA copies of any DNA inserted at the polylinker.
Figure 12.15 A typical expression vector for the synthesis of a hybrid protein.
What if you don't have enough DNA for colony hybridization or Southern blots?
The small sample of DNA can serve as template for DNA polymerase Make complementary primers; add DNA polymerase Add primers in more than 1000-fold excess Heat to separate dsDNA stran ds, then cool Run DNA polymerase (usually Taq) reaction again Repeat heating, cooling, polymerase cycle