principles of biochemistry fourth edition chapter 19 nucleic acids copyright © 2006 pearson...
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Principles of BiochemistryFourth Edition
Chapter 19Nucleic Acids
Copyright © 2006 Pearson Prentice Hall, Inc.
Horton • Moran • Scrimgeour • Perry • Rawn
1869 “ nuclein”Friedrich Miescher treated white blood cells with hydrochloric acid, a precipitate formed that contained carbon, hydrogen, oxygen, nitrogen, and a high percentage of phosphorus.
Nucleic acid.
Hoppe-Seyler isolated ribonucleic acid yeast cells.
1944 Oswald Avery, Colin MacLeod, and Maclyn McCarty demonstrated that DNA is the molecule that carries genetic information.
1953 James D. Watson and Francis H. C. Crick proposed their model of the structure of double-stranded DNA, based on X-ray diffraction patterns that Rosalind Franklin and Maurice Wilkins obtained from DNA fibers, and on the chemical equivalencies noted by Chargaff.
Discovery of DNA
Genome: information resides of the organism. composed of DNA or RNAconsist of a single molecule of DNA in many species of bacteriaconsist of a complete set of DNA molecules found in the nucleus (i.e., the haploid set of chromosomes in diploid organisms) in eukaryotesnot include mitochondrial and chloroplast DNA.
The biological information stored in a cell’s DNA flows from DNA to RNA to protein.
Nucleotides Are the Building Blocks of Nucleic Acids
five-carbon sugar
nitrogenous base
at least one phosphate group
Chemical structures of the two sugars found in nucleotides.
The 2′-endo conformation of deoxyribose predominates in double-stranded DNA.
Chemical structures of pyrimidine and purine.
Pyrimidine has a single ring containing 4 carbon and 2 nitrogen atoms. Purine has a fused pyrimidine-imidazole ring system. Both types of bases are unsaturated, with conjugated double bonds.
Tautomerization reactions
lactim (enol) forms
amino formsimino forms
lactam (keto) forms
Hydrogen bond sites of bases in nucleic acids
hydrogen donors
hydrogen acceptors
Chemical structures of nucleosides
Syn and anti conformations of adenosine
more stable in pyrimidine nucleosides
Chemical structures of the deoxyribonucleoside- 5'-monophosphates.
Deoxyguanosine-5'-monophosphate
DNA Is Double-Stranded
1950 it was clear that DNA is a linear polymer of 2′-deoxyribonucleotide residues linked by 3′-5′ phosphodiesters.Chargaff observed that in the DNA of a given cell, A and T are present in equimolar amounts.
Chemical structure of the tetranucleotide pdApdGpdTpdC(AGTC)
Double-stranded DNA
The two strands run in opposite directions
Three-dimensional structure of B-DNA
Right-hand helixDiameter: 2.37 nmRise: distance between base pairs is 0.33 nm. Pitch: distance to complete one turn is 3.40 nm (10.4 base).
B-DNA
Weak Forces Stabilize the Double Helix
1. Stacking interactions. The stacked base pairs form van der Waals contacts.
2. Hydrogen bonds. Hydrogen bonding between base pairs is a significant stabilizing force.
3. Hydrophobic effects. Burying hydrophobic purine and pyrimidine rings in the interior of the double helix increases the stability of the helix.
4. Charge-charge interactions. Negatively charged phosphate groups of the backbone is a potential source of instability in the DNA helix. However, repulsion is minimized by the presence of cations such as Mg 2+ and cationic proteins (proteins that contain an abundance of the basic residues arginine and lysine)
Complete unwinding and separation of the complementary single strands is called denaturation. Denaturationoccurs only in vitro.Denatured DNA absorbs 12% to 40% more ultraviolet light than doublestranded DNA
Melting curve for DNA
Tm: melting point
A-DNA B-DNA Z-DNA
11 bp/turnRight-hand helixDiameter: 2.37 nmRise: distance between base pairs is 0.33 nm. Pitch: distance to complete one turn is 3.40 nm (10.4 base).2′-endo conformation Anti-conformation of base.
Left-hand helix3′-endo conformation Syn conformation of base
Conformations of Double-Stranded DNA
Supercoiled DNA
Each supercoil compensates for one turn of the double helix.Most circular DNA molecules are supercoiled in cells but even long, linear DNA molecules contain locally supercoiled regions.Most of the DNA in a cell is negatively supercoiled.
Human (Homo sapiens) topoisomerase I bound to DNA
responsible for adding and removing supercoils
1. Ribosomal RNA (rRNA) molecules are an integral part of ribosomes (intracellularribonucleoproteins that are the sites of protein synthesis). Ribosomal RNA is the most abundant class of ribonucleic acid, accounting for about 80% of the total cellular RNA.
2. Transfer RNA (tRNA) molecules carry activated amino acids to the ribosomes for incorporation into growing peptide chains during protein synthesis. tRNA molecules are only 73 to 95 nucleotide residues long. They account for about 15% of the total cellular RNA.
3. Messenger RNA (mRNA) molecules encode the sequences of amino acids in proteins. They are the “messengers” that carry information from DNA to the translation complex where proteins are synthesized. In general, mRNA accounts for only 3% of the total cellular RNA. These molecules are the least stable of the cellular ribonucleic acids.
4. Small RNA molecules are present in all cells. Some small RNA molecules have catalytic activity or contribute to catalytic activity in association with proteins. Many of these RNA molecules are associated with processing events that modify RNA after it has been synthesized.
Cells Contain Several Kinds of RNA
Stem–loop structures in RNA
RNAs are single-stranded molecules with complex secondary structure.
Most single-stranded polynucleotides fold back on themselves to form stable regions of base-paired, double-strandedRNA, such as stem–loop.
DNA Is Packaged in Chromatin in Eukaryotic Cells
chromatin, from the Greek chroma, meaning “color” 1879, Walter Flemming observed banded objects in the nuclei of stained eukaryotic cells.
In a normal resting cell, chromatin exists as long, slender threads about 30 nm in diameter, called 30 nm fibers. In humans, the nucleus must accommodate 46 such chromatin fibers, or chromosomes. The largest human chromosome is about 2.4 * 108 bp, 8 cm long stretched out in the B conformation. During metaphase (when chromosomes are most condensed), chromosome is about 10 um long. In the metaphase chromosome and the extended B form of DNA is 8000-fold (packing ratio).
NucleosomesThe major proteins of chromatin are known as histones.
Except for H1, the amino acid sequence of each type of histone is highly conserved in all eukaryotes. Bovine histone H4 differs from pea histone H4 in only two residues out of 102.
Electron micrograph of extended chromatin showing the “beads-on-a-string” organization.
“ beads”: DNA–histone complexes, nucleosomes “string”: double-stranded DNA
Each nucleosome is composed of a core particle plus histone H1 and linker DNA.The nucleosome core particle is composed of a histone octamer and about 146 bp of DNA. Linker DNA consists of about 54 bp. Histone H1 binds to the core particle and to linker DNA.
Diagram of nucleosome structure.
Each nucleosome is composed of a core particle plus histone H1 and linker DNA.The nucleosome core particle is composed of a histone octamer and about 146 bp of DNA. Linker DNA consists of about 54 bp. Histone H1 binds to the core particle and to linker DNA.
Diagram of nucleosome structure.
Structure of the chicken (Gallus gallus) nucleosome core particle
(a) Histone octamer. (b) Histone octamer bound to DNA
A model of the 30 nm chromatin fiber.
The nucleosomes associate through contacts between adjacent histone H1 molecules.
Electron micrographs of a histone-depleted chromosome
entire protein scaffold
Electron micrographs of a histone-depleted chromosome
Histones Can Be Acetylated and Deacetylated
Amino acid sequence of the N-terminus of histone H4.
Not expressed
Expressed
Nuclease cleavage sites
Exonucleases act on one free end of a polynucleotide and cleave the next phosphodiester linkage.
Endonucleases cleave internal phosphodiester linkages. Cleavage at bond A generates a 5'-phosphate and a 3'-hydroxyl terminus. Cleavage at bond B generates a 3'-phosphate and a 5'-hydroxyl terminus.
Both DNA and RNA are substrates of nucleases (DNase and RNase).
Alkaline hydrolysis of RNA
Step 1, a hydroxide ion abstracts the proton from the 2'-hydroxyl group of a nucleotide residue. The resulting 2'-alkoxide is a nucleophile that attacks the adjacent phosphorus atom, displacing the 5'-oxygen atom and generating a 2',3'-cyclic nucleoside monophosphate.
Step 2, the cyclic intermediate is not stable in alkaline solution, however, and a second hydroxide ion catalyzes its conversion to either a 2'- or 3'-nucleoside monophosphate.
B represents a purine or pyrimidine base.
Mechanism of RNA cleavage by RNase A
Mechanism of RNA cleavage by RNase A
In step 1, His-12 abstracts a proton from the 2'-hydroxyl group of a pyrimidine nucleotide residue. The resulting nucleophilic oxygen atom attacks the adjacent phosphorus atom. His-119 (as an imidazolium ion) donates a proton to the 5'-oxygen atom of the next nucleotide residue to produce an alcohol leaving group,
Mechanism of RNA cleavage by RNase A
Step 2 produces a 2',3'-cyclic nucleoside monophosphate. Water enters the active site on departure of P1.
Mechanism of RNA cleavage by RNase A
In step 3, His-119 (now in its basic form) removes a proton from water. The resulting hydroxide ion attacks the phosphorus atom to form a second transition state
Mechanism of RNA cleavage by RNase A
In step 4, the imidazolium form of His-12 donates a proton to the 2'-oxygen atom, producing P2.
Methylation and restriction at the EcoRI site.
EcoRI bound to DNA
Side view Top view
Restrictionmap of bacteriophage
DNA fingerprinting