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Unformatted text preview: Concepts in Biochemistry
3rd Edition Chapter Ten
DNA and RNA: Structure and Function PART II Dr J. Davis
Chapter Seven Outline
10.1 RNA and DNA Chemical Structures 10.2 DNA Structural Elements 10.3 RNA Structural Elements 10.4 Cleavages of DNA and RNA by Nucleases 10.5 Nucleic Acid-Protein Complexes 2 Chapter 10 Homework
#1 (as needed), 2, 6, 9, 12, 16, 19 21, 29, 31 and 32. Chapter Seven 10.2 DNA 3-D Structural Elements
Tertiary Structure of DNA Quadruplex DNA There is evidence for four-stranded DNA forms aka Quadraplex DNA. Quadraplex DNA [Figure 10.15] has been observed in a protozoan and in a human oncogene. Quadraplex aka G-quadruplexes occurs in Guanine-rich areas of DNA. These structures may play a role in the regulation and stabilizing of telomeres [Chromosome ends], and in regulation of gene expression. Additionally they may limit/ inhibit telomerase action in continuously extending chromosome ends and thus preventing the start of 4 cancer. Figure 10.15 Quadruplex DNA 5 10.4 Cleavage of DNA/RNA Molecules by Nuclease (Enzymes) Biochemists study nucleic acid structures and function by breaking these long, complex molecules into smaller fragments, for: 1) structural analysis of DNA/RNA, and 2) for cleavage at specific sites for recombinant DNA manipulation. Nucleic acid fragmentation procedures include hydrolysis reactions catalyzed by: 1) acids, 2) bases, and 3) by Enzymes. 1. RNA is resistant to hydrolysis by dilute acids; DNA is degraded by 1M HCl by removal of purine bases. 2. DNA is resistant to alkaline [base] hydrolysis; while RNA is easily hydrolyzed with bases, forming polynucleotide fragments of various lengths. 6 10.4 Cleavage of DNA/RNA Molecules by Nuclease (Enzymes) 3. Enzymes are the reagent of choice for careful manipulation, and study of nucleic acid structure. Enzymes allow for cleavage under mild conditions and provide selective, predictable and reproducible cuts in the DNA/RNA chains? Nucleases are enzymes w/i cells that catalyze the hydrolysis of phosphodiester bonds. Nucleases function to remove/degrade damaged or aged nucleic acids (for cellular housekeeping).
7 10.4 Cleavage of DNA/RNA Molecules by Nuclease (Enzymes) Nucleases have specific functions: some work on both DNA & RNA; others are specific for either DNA (Deoxyribonuleases [DNases]) or RNA (Ribonucleases [RNases]). Nucleases isolated and purified from bacterial cells, etc., can be used to perform careful and specific fragmentation of DNA/RNA in the lab. Exonucleases/Endonucleases Exonucleases catalyze hydrolysis of phosphodiester bonds for removal of terminal nucleotides (end).
8 10.4 Cleavage of DNA/RNA Molecules by Nuclease (Enzymes)
Exonucleases/Endonucleases (2) There are two types of Exonucleases (depending on cleavage site): 5'- Exonucleases and 3'- Exonucleases Endonucleases hydrolyze internal phosphodiester bonds. There are two types of internal ester bonds hydrolyzed by Endonucleases (designated type a and type b). Type a bonds connecting the 3'-hydroxyl group and the PO4 group; and Type b bonds connection 5'hydroxyl group and the PO4 group [See Figure 10.21].
9 Fig 10.21 Specificity of Endonucleases 10 Venom from Diamondback rattle snake contains both RNA & DNA nucleases 11 10.4 Cleavage of DNA/RNA Molecules by Nuclease (Enzymes) Exonucleases/Endonucleases The most studied standard nucleases are: rattlesnake venom phosphodiesterase; spleen phosphodiesterase; pancreatic ribonuclease A, and spleen deoxyribonuclease II. Properties for several nucleases are shown in Table 10.5 Note: These enzymes are useful for cutting DNA/RNA into fragments, but do not have selective base specificity.
12 13 10.4 Cleavage of DNA/RNA Molecules by Nuclease (Enzymes)
DNA Restriction Enzymes For specific enzymatic cleavage of DNA, biochemists use Restriction Endonucleases aka Restriction Enzymes [discovered in 1970s]. Bacteria produce these enzymes to degrade any foreign DNA which gets into their cells. Restriction Enzymes (in bacteria) recognize specific base sequence in the foreign double-strand DNA, and hydrolyzes cleavage of the 2 strands near this site. Host DNA (bacterial) is protected from hydrolysis since their bases are methylated.
14 10.4 Cleavage of DNA/RNA Molecules by Nuclease (Enzymes) Several hundred Restriction Enzymes have been isolated (bacteria), characterized, and are sold commercially. The naming of Restriction Enzymes involves: (1) a three letter abbreviation for the source; (2) a letter representing the source strain, and (3) a roman numeral for the order of discovery. Example: EcoRI is the 1st restriction enzyme isolated from E.coli [strain R]. The specificity of EcoRI is shown below (next slide). The cleavage site is a specific hexanucleotide sequence called a palindrome.
15 10.4 Cleavage of DNA/RNA Molecules by Nuclease (Enzymes) Palindromes are sequences that read the same in the 5' to 3' direction. The specificity of Restriction Enzyme, EcoRI (5'GAATTC): Two phosphodiester bonds are hydrolyzed (red arrows), fragmenting both strands of DNA. 16 10.4 Cleavage of DNA/RNA Molecules by Nuclease (Enzymes) The specificity of Restriction Enzyme, EcoRI: Once separated, each of the 2 cleaved strands has a single stranded (ss) 5' pAATT end. These ss tails are called cohesive ends. They can be used to insert new DNA sequence into a DNA chain (Later). 17 10.4 Cleavage of DNA/RNA Molecules by Nuclease (Enzymes) Other restriction endonucleases (SmaI) generate blunt ends by cutting in the middle of a palindrome (5'-CCCGGG). Several DNA Restriction Enzymes are listed on the bottom of Table 10.5, including EcoRI; TaqI, and HinfI, etc. Several hundred of these enzymes have now been discovered. The specificities of several other important RE are shown in the handouts. BamHI and HindIII (like EcoRI) also perform staggered cleavage, leaving a single strand of DNA ("sticky-ends"). 18 19 10.4 Cleavage of DNA/RNA Molecules by Nuclease (Enzymes)
Uses of Restriction Endonucleases Nucleases are used extensively in molecular biology & Biochemistry (hundreds are available for purchase commercially). Restriction enzymes cut large DNA molecules into smaller fragments so they can be more easily analyzed. The sites of cleavage of several DNA restriction enzymes acting on Lambda phage (bacteriophage) DNA are shown in Figure 10.22.
20 10.4 Cleavage of DNA/RNA Molecules by Nuclease (Enzymes)
Figure 10.22 Restriction enzyme cleavage sites on phage DNA for 2 restriction enzymes (EcoRI & HpaI). 21 10.4 Cleavage of DNA/RNA Molecules by Nuclease (Enzymes)
Uses of Restriction Endonucleases Lambda () Phage DNA is a linear, ds-DNA molecule with ~48,500 base pairs. EcoRI would cut this DNA into 6 fragments; while HpaI (RE from a parainfluenza bacteria) makes 15 cuts. The base sequence recognized by each RE only occurs a few times per molecule. Larger DNA molecules (rodents/primates) would produce many sites for fragmentation yielding 100's of fragments. For smaller DNA molecules, there is a good chance that we can produce an unique set of fragments, giving a unique fingerprint of the DNA structure. 22 10.4 Cleavage of DNA/RNA Molecules by Nuclease (Enzymes)
Uses of Restriction Endonucleases Fingerprints are readily separated and sized by agarose gel electrophoresis (see extra slides). RNA Endonucleases???? There are no analogous enzymes to cleave RNA into well-defined fragments. A new discovery: Ribozymes (catalytic RNA) are being explored as potential RNA restriction endonucleases. Restriction enzymes in Forensics/Anthropology, etc. Restriction enzyme cleavage is now widely applied in Forensic chemistry/biology for analysis of crime scene DNA evidence in body fluids, etc. See extra slides. 23 10.5 NUCLEIC ACID-PROTEIN COMPLEXES Recall: Supramolecular Assemblies [Chap 1] are organized clusters of macromolecules that perform specific biochemical functions. NUCLEOPROTEIN Complexes are examples of such molecules that contain combined assemblies of proteins and nucleic acids. Examples include: (1) Viruses stable, infective particles composed of nucleic acids, DNA or RNA and protein subunits. (2) Bacteriophages (aka PHAGES) viruses specific for bacteria. Most phages are DNA viruses. Example: X174 which infects E.coli. This phage has only 5400 nucleotide bases in its genome. 24 10.5 NUCLEIC ACID-PROTEIN COMPLEXES (3) Plant Viruses include the tobacco mosaic virus (TMV)
which is a ss-RNA virus [~ 6390 nucleotides]. (4) Animal Viruses TE hundreds of animal viruses; some highly pathogenic to humans. Many are RNA viruses, which requires an enzyme called Reverse Transcriptase [RT] to synthesize a DNA complementary to the viral RNA genome. HIV is an example: It is a retrovirus containing 2 copies of ss-RNA in its genome [~ 9700 nucleotides]
25 10.5 NUCLEIC ACID-PROTEIN COMPLEXES
HIV Reverse Transcriptase (RT) (cont.) Reverse transcriptase (RT) copies or transforms its RNA into a ds DNA form that then incorporates into the hosts cells' genome. When the host T-cell starts to replicate itself it also creates thousands of new viruses which destroy/kill the cell [human T cell]. HIV has 2 critical membrane glycoproteins, gp41 and gp121 (green: Fig 10.23) which are constantly changing making a vaccine difficult to develop. Recall: DDI, AZT, DDC and others have been developed to inhibit Reverse transcriptase.
26 Fig 10.23 Schematic Diagram of HIV 27 10.4 Cleavage of DNA/RNA Molecules by Nuclease (Enzymes)
Chromosomes Genomic DNA is localized in the cell nucleus in functional units called Chromosomes. In mammals, chromosomes consists of packages with DNA and Protein. Chromosomes are highly ordered and compact linear molecules = ~1-2 meters long [Note: the nucleus is only ~5 m in diameter]. AAaaastounding!!! CHROMATIN is the name given for the entire nucleoprotein complex (Eukaryotes), consisting of DNA molecules plus two classes of proteins: a) Histones and b) Nonhistone Chromosomal proteins.
28 10.4 Cleavage of DNA/RNA Molecules by Nuclease (Enzymes) Chromosomes Histones are small basic proteins with lots of Arginine and Lysine aa residues [size: 10 20, 000 daltons]. Wrapping the genetic material into a package small enough to fit them into the cell nucleus requires a wellorganized and compact configuration/arrangement of DNA molecules. The packaging of DNA into chromosomes is illustrated in Figure 10.24. The packaging process starts with DNA coiled around histone protein cores (aka Nucleosomes).
29 10.4 Cleavage of DNA/RNA Molecules by Nuclease (Enzymes) Chromosomes There are ~ millions of nucleosomes around which the DNA is wrapped ["beads on a string"]. Next, several Nucleosomes arrange into a chromatin fiber, which are further wrapped into single loops, and then into multiple structures with 6 loops (called Rosettes), and finally into grouping of 30 rosettes (called coils), and finally into loop clusters (called Chromatids) Chromatids combine to form the final chromosome unit. Process allows for compacting the DNA molecule into structures that are about ~1/100, 000x original volume. 30 Figure 10.24 DNA in Chromosomes 31 32 10.4 Cleavage of DNA/RNA Molecules by Nuclease (Enzymes)
snRNPs A new class of ribonucleoprotein complexes that play a role in RNA processing, aka small nuclear Ribonucleoprotein particles (snRNPs = snurps). These small particles [100 - 200 nucleotide bases], play an important role in preparation/processing of mRNA. They catalyze specific splicing reactions that transform gene transcripts [heterogeneous nuclear RNA] into mature mRNA, before transport from the nucleus to the ribosomes in the cytoplasm. They remove intron regions (noncoding) from mRNA leaving the coding sequences [Exons] which are joined into active mRNA. 33 10.4 Cleavage of DNA/RNA Molecules by Nuclease (Enzymes)
RIBOSOMES Ribosomes are supramolecular assemblies of RNA and Protein that function as intracellular sites for protein synthesis [translation of RNA]. Recall: Ribosomes consist of ~65% RNA and ~35% Protein. Ribonucleoprotein Enzymes Ribonuclease P, Telomerase and Peptidyl transferase are important biomolecules which function as enzymes but are composed of protein and nucleic acids parts.
34 10.4 Cleavage of DNA/RNA Molecules by Nuclease (Enzymes) There are, however, two examples of RNA molecules acting as enzymes: 1) Ribozymes, and 2) Self-splicing Introns. 1. Ribozymes are RNA molecules that can act like enzymes (discovered in the 1980s [Noble prize awarded to Sid Altman and Tom Cech in 1989]). Ribozymes (aka Catalytic RNA) are under development for use in elucidating RNA structure; they may also find use as new pharmaceuticals/drugs that can potentially control viral and bacterial infections. Examples of Ribozymes: Ribonuclease P Ribonuclease P molecule consists of a 14 kd protein plus a 377 nucleotide RNA in a single chain. 35 10.4 Cleavage of DNA/RNA Molecules by Nuclease (Enzymes) Dogma: Most known enzymes are proteins!!!. The catalytic properties for Ribonuclease P molecule, however, reside in the RNA part, which obeys MM kinetics etc. A potential commercial use for ribozymes, might be to perform in vivo cleavage and /or inactivation of non-host RNA inside cells infected with viruses, retroviruses (or even certain cancers ???).
Self-splicing Introns rRNA has been shown to be able to splice out sections of its own structure w/o protein involvement. Other studies indicate that these molecules can also perform cleavage and rejoining of nucleotides, the hydrolysis of phosphodiester bonds, and many other bizarre activities.
36 End of Chapter 10 Chapter Seven ...
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