10.5 What Are the Different Classes of Nucleic Acids? 303 histone chromosomal proteins, many of which are involved in regulating which genes in DNA are transcribed at any given moment. The amount of DNA in a diploid mammalian cell is typically more than 1000 times that found in an E. coli cell. Some higher plant cells contain more than 50,000 times as much. Various Forms of RNA Serve Different Roles in Cells Unlike DNA, cellular RNA molecules are almost always single-stranded. However, all of them typically contain double-stranded regions formed when stretches of nu- cleotides with complementary base sequences align in an antiparallel fashion and form canonical AϺU and GϺC base pairs. (Compare Figures 10.3 and 10.17 to con- vince yourself that U would pair with A in the same manner T does.) Such base pair- ing creates secondary structure. Messenger RNA Carries the Sequence Information for Synthesis of a Protein Messenger RNA (mRNA) serves to carry the information or “message” that is en- coded in genes to the sites of protein synthesis in the cell, where this information is translated into a polypeptide sequence. That is, mRNA molecules are transcribed copies of the protein-coding genetic units of DNA. Prokaryotic mRNAs have from 75 to 3,000 nucleotides; mRNA constitutes about 2% of total prokaryotic RNA. Messenger RNA is synthesized during transcription, an enzymatic process in which an RNA copy is made of the sequence of bases along one strand of DNA. This mRNA then directs the synthesis of a polypeptide chain as the information that is contained within its nucleotide sequence is translated into an amino acid sequence by the protein-synthesizing machinery of the ribosomes. Ribosomal RNA and tRNA mole- cules are also synthesized by transcription of DNA sequences, but unlike mRNA mol- ecules, these RNAs are not subsequently translated to form proteins. In prokaryotes, a single mRNA may contain the information for the synthesis of several polypeptide chains within its nucleotide sequence (Figure 10.20). In contrast, eukaryotic mRNAs encode only one polypeptide but are more complex in that they are synthesized in the nucleus in the form of much larger precursor molecules called heterogeneous nuclear RNA, or hnRNA. hnRNA molecules contain stretches of nucleotide sequence that have no protein-coding capacity. These noncoding regions are called intervening sequences or introns because they intervene between coding regions, which are called exons. Introns interrupt the continuity of the information specifying the amino acid sequence of a protein and must be spliced out before the message can be translated. In addition, eukaryotic hnRNA and mRNA molecules have a run of 100 to 200 adenylic acid residues attached at their 3Ј-ends, so-called poly(A) tails. This poly- adenylation occurs after transcription has been completed and is essential for effi- cient translation and stability of the mRNA. The properties of mRNA molecules as they move through transcription and translation in prokaryotic versus eukaryotic cells are summarized in Figure 10.20. Ribosomal RNA Provides the Structural and Functional Foundation for Ribosomes Ribosomes, the supramolecular assemblies where protein synthesis occurs, are about 65% RNA of the ribosomal RNA type. Ribosomal RNA (rRNA) molecules fold into characteristic secondary structures as a consequence of intramolecular base-pairing interactions (Figure 10.21). The different species of rRNA are generally referred to according to their sedimentation coefficients 1 (see the Appendix to Chapter 5), which are a rough measure of their relative size (Figure 10.22). Ribosomes are composed of two subunits of different sizes that dissociate from each other if the Mg 2ϩ concentration is below 10 Ϫ3 M. Each subunit is a supramol- ecular assembly of proteins and RNA and has a total mass of 10 6 D or more. E. coli ribosomal subunits have sedimentation coefficients of 30S (the small subunit) and 50S (the large subunit). Eukaryotic ribosomes are somewhat larger than prokary- 1 Sedimentation coefficients are a measure of the velocity with which a particle sediments in a cen- trifugal force field. Sedimentation coefficients are expressed in Svedbergs (symbolized S), named to honor The Svedberg, developer of the ultracentrifuge. One Sϭ10 Ϫ13 sec. 304 Chapter 10 Nucleotides and Nucleic Acids otic ribosomes, consisting of 40S and 60S subunits. More than 80% of total cellular RNA is represented by the various forms of rRNA. Ribosomal RNAs characteristically contain a number of specially modified nu- cleotides, including pseudouridine residues, ribothymidylic acid, and methylated bases (Figure 10.23). The central role of ribosomes in the biosynthesis of proteins is treated in detail in Chapter 30. Here we briefly note the significant point that ge- netic information in the nucleotide sequence of an mRNA is translated into the amino acid sequence of a polypeptide chain by ribosomes. Transfer RNAs Carry Amino Acids to Ribosomes for Use in Protein Synthesis Transfer RNAs (tRNAs) serve as the carrier of amino acids for protein synthesis (see Chapter 30). tRNA molecules also fold into a characteristic secondary structure (Figure 10.24). tRNAs are small RNA molecules, containing 73 to 94 residues, a sub- stantial number of which are methylated or otherwise unusually modified. Each of the 20 amino acids in proteins has at least one unique tRNA species dedicated to chauffeuring its delivery to ribosomes for insertion into growing polypeptide chains, and some amino acids are served by several tRNAs. In eukaryotes, there are even discrete sets of tRNA molecules for each site of protein synthesis—the cyto- plasm, the mitochondrion, and in plant cells, the chloroplast. All tRNA molecules possess a 3Ј-terminal nucleotide sequence that reads -CCA, and the amino acid is FIGURE 10.21 Ribosomal RNA has a complex secondary structure due to many intrastrand hydrogen bonds.The gray line in this figure traces a polynucleotide chain con- sisting of more than 1000 nucleotides. Aligned regions represent H-bonded complementary base sequences. Prokaryotes: 5'DNA segment 3' Gene A Gene B Gene C RNA polymerase 5' mRNA encoding proteins A, B, C Ribosome A polypeptide A protein B polypeptide B protein C polypeptide DNA-dependent RNA polymerase transcribing DNA of genes A, B, C Ribosomes translating mRNA into proteins A, B, C Eukaryotes: Exons are protein-coding regions that must be joined by removing introns, the noncoding intervening sequences. The process of intron removal and exon joining is called splicing. DNA segment 3' Gene A Exon 1 Intron Exon 2 5' Transcription DNA transcribed by DNA-dependent RNA polymerase Exon 1 Intron Exon 2 hnRNA (encodes only one polypeptide) 5'–untranslated region 3'–untranslated region AAAA Poly(A) added after transcription Splicing Transport to cytoplasm 5'mRNA snRNPs Exon 1 Exon 2 3'AAAA Translation mRNA is transcribed into a protein by cytoplasmic ribosomes Protein A mRNA ANIMATED FIGURE 10.20 Transcription and translation of mRNA mole- cules in prokaryotic versus eukaryotic cells. See this figure animated at www.cengage .com/login. 10.5 What Are the Different Classes of Nucleic Acids? 305 carried to the ribosome attached as an acyl ester to the free 3Ј-OH of the terminal A residue. These aminoacyl-tRNAs are the substrates of protein synthesis, the amino acid being transferred to the carboxyl end of a growing polypeptide. The peptide bond–forming reaction is a catalytic process intrinsic to ribosomes. Small Nuclear RNAs Mediate the Splicing of Eukaryotic Gene Transcripts (hnRNA) into mRNA Small nuclear RNAs, or snRNAs, are a class of RNA molecules found in eukaryotic cells, principally in the nucleus. They are neither tRNA nor small rRNA molecules, although they are similar in size to these species. They contain from 100 to about 200 nucleotides, some of which, like tRNA and rRNA, are methy- lated or otherwise modified. No snRNA exists as naked RNA. Instead, snRNA is found in stable complexes with specific proteins forming small nuclear ribonucleo- protein particles, or snRNPs, which are about 10S in size. Their occurrence in eu- karyotes, their location in the nucleus, and their relative abundance (1% to 10% of the number of ribosomes) are significant clues to their biological purpose: snRNPs are important in the processing of eukaryotic gene transcripts (hnRNA) into ma- ture messenger RNA for export from the nucleus to the cytoplasm (Figure 10.20). PROKARYOTIC RIBOSOMES (E. coli) EUKARYOTIC RIBOSOMES (Rat) Ribosome (2.52 ϫ 10 6 D) 70S Subunits 30S 50S (0.93 ϫ 10 6 D) (1.59 ϫ 10 6 D) RNA 16S RNA (1542 nucleotides) 23S RNA (2904 nucleotides) 5S RNA (120 nucleotides) Protein 21 proteins 31 proteins Ribosome (4.22 ϫ 10 6 D) Subunits 40S 60S (1.4 ϫ 10 6 D) (2.82 ϫ 10 6 D) RNA 18S RNA (1874 nucleotides) 28S + 5.85 RNA (4718 + 160 nucleotides) 5S RNA (120 nucleotides) Protein 33 proteins 49 proteins 80S FIGURE 10.22 The organization and composition of prokaryotic and eukaryotic ribosomes. S Ribose 4-Thiouridine (S 4 U) H O O Inosine H Ribothymidine (T) H CH 3 Pseudouridine (␺) H Dihydrouridine (D) H H H H N NN N N N O H N O Ribose H H O H N O N H Ribose H O H N N O Ribose N Ribose FIGURE 10.23 Unusual bases in RNA. 3' 5' FIGURE 10.24 Transfer RNA also has a complex secondary structure due to many intrastrand hydrogen bonds.The black lines represent base-paired nucleotides in the sequence. 306 Chapter 10 Nucleotides and Nucleic Acids Small RNAs Serve a Number of Roles, Including Gene Silencing A class of RNA molecules even smaller than tRNAs is the small RNAs, so-called because they are only 21 to 28 nucleotides long. (Some refer to this class as the noncoding RNAs [or ncRNAs]. Others refer to small RNAs as regulatory RNAs, because virtually every step along the pathway of gene expression can be regulated by one or another small RNA.) Small RNAs are involved in a number of novel biological functions. These small RNAs can target DNA or RNA through complementary base pairing. Base pairing of the small RNA with particular nucleotide sequences in the target is called direct readout. Small RNAs are classified into a number of subclasses on the basis of their function. RNA interference (RNAi) is mediated by one subclass, the small inter- fering RNAs (siRNAs). siRNAs disrupt gene expression by blocking specific protein production, even though the mRNA encoding the protein has been syn- thesized. The 21- to 23-nucleotide-long siRNAs act by base pairing with comple- mentary sequences within a particular mRNA to form regions of double-stranded RNA (dsRNA). These dsRNA regions are then specifically degraded, eliminating the mRNA from the cell (see Chapter 12). Thus, RNAi is a mechanism to silence the expression of specific genes, even after they have been transcribed, a phe- nomenon referred to as gene silencing. RNAi is also implicated in modifying the structure of chromatin and causing large-scale influences in gene expression. An- other subclass, the micro RNAs (miRNAs) control developmental timing by base pairing with and preventing the translation of certain mRNAs, thus blocking syn- thesis of specific proteins. Thus, miRNAs also act in gene silencing. However, un- like siRNAs, miRNAs (22 nucleotides long) do not cause mRNA de gradation. A third subclass is the small nucleolar RNAs (snoRNAs). snoRNAs (60 to 300 nu- cleotides long) are catalysts that accomplish some of the chemical modifications A DEEPER LOOK The RNA World and Early Evolution Proteins are encoded by nucleotide sequences in DNA. DNA repli- cation depends on the activity of protein enzymes. These two state- ments form a “chicken and egg” paradox: Which came first in evolution—DNA or protein? Neither, it seems. The 1989 Nobel Prize in Chemistry was awarded to Thomas Cech and Sidney Altman for their discovery that RNA molecules are not only informational but also may be catalytic. This discovery gave evidence to earlier specu- lation by Carl Woese, Francis Crick, and Leslie Orgel that prebiotic evolution (that is, early evolution before cells arose) depended on self-replicating and catalytic RNAs, with proteins and DNA appear- ing later. Three basic assumptions about the prebiotic RNA world are (1) RNA replication maintained information-carrying RNAs, (2) Watson–Crick base pairing was essential to RNA replication, and (3) genetically encoded proteins were unavailable as catalysts. The challenge shifts to explaining the origin of nucleotides and their polymerization to form RNA. Adenine exists in outer space and is found in comets and me- teorites. A likely source is conversion of aminoimidazolecarboni- trile to adenine. (Aminoimidazolecarbonitrile is a tetramer of HCN; adenine is a pentamer.) Glycolaldehyde can combine with other simple compounds to form ribose (and glucose). Glycolaldehyde has been detected in a gas cloud at the center of the Milky Way, our galaxy. (Acetic acid and methyl formate have the same eight atoms as gly- colaldehyde; these two useful precursor molecules have also been detected in intergalactic clouds.) Inorganic phosphate, the re- maining ingredient in nucleotides, is a common component in naturally occurring aqueous solutions. Its negative charge allows it to interact readily with positively charged mineral surfaces, upon which the first nucleotides may have spontaneously assembled. These tantalizing facts are bright spots along the dim thread that connects us to our distant past. The RNA world is an attractive hypothesis. Reference: Gesteland, R. F., Cech, T. R., Atkins, J. F., eds., 2006. The RNA World: The Nature of Modern RNA Suggests a Prebiotic RNA World, 3rd ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press. CO C H OH C CC H H H H CH 2 OH OH OH HO D-Ribose C 5 H 10 O 5 HO C COHH C CH 2 OH OHH COHH Glycolaldehyde C 2 H 4 O 2 ␣-D-Glucopyranose C 6 H 12 O 6 HO C CH 2 OH Adenine N N NH 2 N N H Aminoimidazolecarbonitrile H 2 N C N N N H ᮡ Adapted from Glaser, R., et al., 2007. Adenine synthesis in interstellar space: Mechanisms of prebiotic pyrimidine-ring formation of monocyclic HCN-pentamers. Astrobiology 7:455–470. 10.6 Are Nucleic Acids Susceptible to Hydrolysis? 307 found in tRNA, rRNA, and even DNA (see Figure 10.23, for example). Small RNAs in bacteria (known by the acronym sRNAs) play an important role altering gene expression in response to stressful environmental situations. The Chemical Differences Between DNA and RNA Have Biological Significance Two fundamental chemical differences distinguish DNA from RNA: 1. DNA contains 2-deoxyribose instead of ribose. 2. DNA contains thymine instead of uracil. What are the consequences of these differences, and do they hold any significance in common? An argument can be made that, because of these differences, DNA is chemically more stable than RNA. The greater stability of DNA over RNA is consis- tent with the respective roles these macromolecules have assumed in heredity and information transfer. Consider first why DNA contains thymine instead of uracil. The key observation is that cytosine deaminates to form uracil at a finite rate in vivo (Figure 10.25). Because C in one DNA strand pairs with G in the other strand, whereas U would pair with A, conversion of a C to a U could potentially result in a heritable change of a CϺG pair to a UϺA pair. Such a change in nucleotide sequence would constitute a mutation in the DNA. To prevent this C deamination from leading to permanent changes in nu- cleotide sequence, a cellular repair mechanism “proofreads” DNA, and when a U arising from C deamination is encountered, it is treated as inappropriate and is re- placed by a C. If DNA normally contained U rather than T, this repair system could not readily distinguish U formed by C deamination from U correctly paired with A. However, the U in DNA is “5-methyl-U” or, as it is conventionally known, thymine (Figure 10.26). That is, the 5-methyl group on T labels it as if to say “this U belongs; do not replace it.” The other chemical difference between RNA and DNA is that the ribose 2Ј-OH group on each nucleotide in RNA is absent in DNA. Consequently, the ubiquitous 3Ј-O of polynucleotide backbones lacks a vicinal hydroxyl neighbor in DNA. This difference leads to a greater resistance of DNA to alkaline hydrolysis, examined in detail in the following section. To view it another way, RNA is less stable than DNA because its vicinal 2Ј-OH group makes the 3Ј-phosphodiester bond susceptible to nucleophilic cleavage (Figure 10.27). For just this reason, it is selectively advantageous for the heritable form of genetic information to be DNA rather than RNA. 10.6 Are Nucleic Acids Susceptible to Hydrolysis? Most reactions of nucleic acid hydrolysis break phosphodiester bonds in the polynu- cleotide backbone even though such bonds are among the most stable chemical bonds found in biological molecules. In the laboratory, hydrolysis of polynu- cleotides will generate smaller fragments that are easier to manipulate and study. RNA Is Susceptible to Hydrolysis by Base, But DNA Is Not RNA is relatively resistant to the effects of dilute acid, but gentle treatment of DNA with 1 mM HCl leads to hydrolysis of purine glycosidic bonds and the loss of purine bases from the DNA. The glycosidic bonds between pyrimidine bases and 2Ј-deoxyribose are not affected, and in this case, the polynucleotide’s sugar– phosphate backbone remains intact. The purine-free polynucleotide product is called apurinic acid. DNA is not susceptible to alkaline hydrolysis. On the other hand, RNA is alkali labile and is readily hydrolyzed by hydroxide ions (Figure 10.27). DNA has no 2Ј-OH; therefore, DNA is alkali stable. Cytosine N N H O NH 2 + H 2 O Uracil N N H O H +NH 3 O FIGURE 10.25 Deamination of cytosine forms uracil. O CH 3 N H O N H 4 5 6 1 2 3 FIGURE 10.26 The 5-methyl group on thymine labels it as a special kind of uracil. 308 Chapter 10 Nucleotides and Nucleic Acids The Enzymes That Hydrolyze Nucleic Acids Are Phosphodiesterases Enzymes that hydrolyze nucleic acids are called nucleases. Virtually all cells contain various nucleases that serve important housekeeping roles in the normal course of nucleic acid metabolism. Organs that provide digestive fluids, such as the pancreas, are rich in nucleases and secrete substantial amounts to hydrolyze ingested nucleic acids. Fungi and snake venom are often good sources of nucleases. As a class, nucle- ases are phosphodiesterases because they catalyze the cleavage of phosphodiester O _ OH P O O O – AC OH P O O O – U OH P O O O – G OH etc. etc. A nucleophile such as OH – can abstract the H of the 2'–OH, generating 2'–O – which attacks the ␦ + P of the phosphodiester bridge: OH P O O O – AC OH P O O O – OH P O O O – U OH etc. OH P O O O – AC P O O O – U OH P O O O – G OH etc. etc. OH P O O O – AC P O O U OH P O O O – G OH etc. etc. OH – G etc. ␦ + O O – HO + Sugar–PO 4 backbone cleaved OOO OOO OOO OO H 2 O H 2 O or C OH PO O – O – 2' 3' 2'-PO 4 product C OH 2' 3' 3'-PO 4 product PO O – O – Complete hydrolysis of RNA by alkali yields a random mixture of 2'-NMPs and 3'-NMPs. O O OH P O O O – A etc. A etc. O OH P O O O – O H 2 O 1 2 12 ANIMATED FIGURE 10.27 Alkaline hydrolysis of RNA.The vertical lines represent ribose; the diagonals the phosphodiester linkages joining succes- sive nucleotides. Nucleophilic attack by OH Ϫ on the P atom leads to 5Ј-phosphoester cleavage and random hydrolysis of the cyclic 2Ј,3Ј-phosphodiester intermedi- ate to give a mixture of 2Ј- and 3Ј-nucleoside mono- phosphate products. See this figure animated at www.cengage.com/login. 10.6 Are Nucleic Acids Susceptible to Hydrolysis? 309 bonds by H 2 O. Because each internal phosphate in a polynucleotide backbone is involved in two phosphoester linkages, cleavage can potentially occur on either side of the phosphorus (Figure 10.28). Convention labels the 3Ј-side as a and the 5Ј-side as b. Enzymes or reactions that hydrolyze nucleic acids are characterized as acting at either a or b. A second convention denotes whether the nucleic acid chain was cleaved at some internal location, endo, or whether a terminal nucleotide residue was hydrolytically removed, exo. Note that exo a cleavage occurs at the 3Ј-end of the poly- mer, whereas exo b cleavage involves attack at the 5Ј-terminus (Figure 10.28). Nucleases Differ in Their Specificity for Different Forms of Nucleic Acid Nucleases play an indispensable role in the cellular breakdown of nucleic acids and salvage of their constituent parts. Nucleases also participate in many other cellular functions, including (1) aspects of DNA metabolism, such as replication and repair; (2) aspects of RNA metabolism, such as splicing of the primary gene transcript, pro- cessing of mRNA, and RNAi; (3) rearrangements of genetic material, such as re- combination and transposition; (4) host defense mechanisms against foreign nucleic acid molecules; and (5) the immune response, through assembly of immunoglobu- lin genes (these topics are discussed in depth in Part IV). Some nucleases are not even proteins but instead are catalytic RNA molecules (see Chapter 13). Like most enzymes (see Chapter 13), nucleases exhibit selectivity or specificity for the nature of the substance on which they act. That is, some nucleases act only on DNA (DNases), whereas others are specific for RNA (the RNases). Still others are nonspecific and are referred to simply as nucleases. Nucleases may also show specificity for only single-stranded nucleic acids or may act only on double helices. Some display a decided preference for acting only at certain bases in a polynucleotide, or as we shall see for restriction endonucleases, act only at a particular nucleotide sequence four to eight nucleotides (or more) in length. To the molecular biologist, nucleases are the surgical tools for the dissection and manipulation of nucleic acids in the laboratory. A P P G C P T P A P OH abababab Convention: The 3'-side of each phosphodiester is termed a ; the 5'-side is termed b . Hydrolysis of the a bond yields 5'-PO 4 products: A OH G OH C OH T OH A OH Mixture of 5'-nucleoside monophosphates (NMPs) Hydrolysis of the b bond yields 3'-PO 4 products: (a) (b) A G HO C HO T HO A HO OH A 3',5'-diPO 4 nucleotide from the 5'-end A mixture of 3'-NMPs A nucleoside from the 3'-OH end P P P P P P P P P P FIGURE 10.28 Cleavage in polynucleotide chains. (a) Cleavage on the a side leaves the phosphate attached to the 5Ј-position of the adjacent nucleotide, while (b) b-side hydrolysis yields 3Ј-phosphate products. 310 Chapter 10 Nucleotides and Nucleic Acids Restriction Enzymes Are Nucleases That Cleave Double-Stranded DNA Molecules Restriction endonucleases are enzymes, isolated chiefly from bacteria, that have the ability to cleave double-stranded DNA. The term restriction comes from the capacity of prokaryotes to defend against or “restrict” the possibility of takeover by foreign DNA that might gain entry into their cells. Prokaryotes degrade foreign DNA by us- ing their unique restriction enzymes to chop it into relatively large but noninfective fragments. Restriction enzymes are classified into three types: I, II, or III. Types I and III require ATP to hydrolyze DNA and can also catalyze chemical modification of DNA through addition of methyl groups to specific bases. Type I restriction en- donucleases cleave DNA randomly, whereas type III recognize specific nucleotide sequences within dsDNA and cut the DNA at or near these sites. Type II Restriction Endonucleases Are Useful for Manipulating DNA in the Lab Type II restriction enzymes have received widespread application in the cloning and sequencing of DNA molecules. Their hydrolytic activity is not ATP-dependent, and they do not modify DNA by methylation or other means. Most important, they cut DNA within or near particular nucleotide sequences that they specifically rec- ognize. These recognition sequences are typically four or six nucleotides in length and have a twofold axis of symmetry. For example, E. coli has a restriction enzyme, EcoRI, that recognizes the hexanucleotide sequence GAATTC: Note the twofold symmetry: the sequence read 5Ј→3Ј is the same in both strands. When EcoRI encounters this sequence in dsDNA, it causes a staggered, double- stranded break by hydrolyzing each chain between the G and A residues: Staggered cleavage results in fragments with protruding single-stranded 5Ј-ends: Because the protruding termini of EcoRI fragments have complementary base se- quences, they can form base pairs with one another. Therefore, DNA restriction fragments having such “sticky” ends can be joined to- gether to create new combinations of DNA sequence. If fragments derived from DNA molecules of different origin are combined, novel recombinant forms of DNA are created. EcoRI leaves staggered 5Ј-termini. Other restriction enzymes, such as PstI, which recognizes the sequence 5Ј-CTGCAG-3Ј and cleaves between A and G, pro- duce cohesive staggered 3Ј-ends. Still others, such as Bal I, act at the center of the twofold symmetry axis of their recognition site and generate blunt ends that are noncohesive. Bal I recognizes 5Ј -TGGCCA-3Ј and cuts between G and C. Table 10.2 lists many of the commonly used restriction endonucleases and their recognition sites. Different restriction enzymes sometimes recognize and cleave NNN N N N N NG AAT T C NNN N N N N NC T T A A G 5Ј NN 3ЈN N N N N NG 5Ј AAT T C 3Ј NN 5ЈN N N N N NC T T A A 5Ј G 5Ј N N 3ЈN N N N N NG AAT T C 3Ј NN 5ЈN N N N N NC T T A A G 5Ј NN 3ЈN N N N N NG A A T T C 3Ј NN 5ЈN N N N N NC T T A A G 10.6 Are Nucleic Acids Susceptible to Hydrolysis? 311 About 1000 restriction enzymes have been characterized. They are named by italicized three-letter codes; the first is a capital letter denoting the genus of the organism of origin, and the next two letters are an abbreviation of the particular species. Because prokaryotes often contain more than one restriction enzyme, the various representatives are assigned letter and number codes as they are identified. Thus, EcoRI is the initial restriction endonuclease isolated from Escherichia coli, strain R. With one exception (NciI), all known type II restriction endonucleases generate fragments with 5Ј-PO 4 and 3Ј-OH ends. Common Recognition Enzyme Isoschizomers Sequence Compatible Cohesive Ends AluIAGgCT Blunt ApyI AtuI, EcoRII CCgG( A T )GG AsuII TTgCGAA ClaI, HpaII, TaqI Ava IGgPyCGPuG SalI, XhoI, XmaI Avr II CgCTAGG Bal I TGGgCCA Blunt BamHI GgGATCC Bcl I, Bgl II, MboI, Sau3A, XhoII Bcl ITgGATCA BamHI, Bgl II, MboI, Sau3A, XhoII Bgl II AgGATCT BamHI, BclI, MboI, Sau3A, XhoII BstEII GgGTNACC BstXI CCANNNNNgNTGG ClaIATgCGAT AccI, AcyI, AsuII, HpaII, TaqI DdeICgTNAG EcoRI GgAATTC EcoRII AtuI, ApyI gCC( A T )GG FnuDII ThaICGgCG Blunt HaeI( A T )GGgCC( T A ) Blunt HaeII PuGCGCgPy HaeIII GGgCC Blunt HincII GTPygPuAC Blunt HindIII AgAGCTT HpaI GTTgAAC Blunt HpaII CgCGG AccI, AcyI, AsuII, ClaI, TaqI KpnI GGTACgC MboI Sau3A gGATC BamHI, Bcl I, Bgl II, XhoII MspICgCGG MstI TGCgGCA Blunt NotIGCgGGCCGC PstI CTGCAgG SacI SstI GAGCTgC Sal IGgTCGAC AvaI, XhoI Sau3A gGATC BamHI, Bcl I, Bgl II, MboI, XhoII SfiI GGCCNNNNgNGGCC SmaI XmaI CCCgGGG Blunt SphI GCATGgC Sst I SacI GAGCTgC Taq ITgCGA AccI, AcyI, AsuII, ClaI, HpaII XbaITgCTAGA XhoICgTCGAG AvaI, SalI XhoII ( A G )gGATC( T C ) BamHI, BclI, Bgl II, MboI, Sau3A XmaI SmaICgCCGGG AvaI TABLE 10.2 Restriction Endonucleases 312 Chapter 10 Nucleotides and Nucleic Acids within identical target sequences. Such enzymes are called isoschizomers, meaning that they cut at the same site; for example, MboI and Sau3A are isoschizomers. Restriction Fragment Size Assuming random distribution and equimolar pro- portions for the four nucleotides in DNA, a particular tetranucleotide sequence should occur every 4 4 nucleotides, or every 256 bases. Therefore, the fragments generated by a restriction enzyme that acts at a four-nucleotide sequence should average about 250 bp in length. “Six-cutters,” enzymes such as EcoRI or BamHI, will find their unique hexanucleotide sequences on the average once in every 4096 (4 6 ) bp of length. Because the genetic code is a triplet code with three suc- cessive bases in a DNA strand specifying one amino acid in a polypeptide sequence, and because polypeptides typically contain at most 1000 amino acid residues, the fragments generated by six-cutters are approximately the size of prokaryotic genes. This property makes these enzymes useful in the construction and cloning of ge- 1 2 3 4 5 6 7 8 1 5 2 7 1 5 2 7 ABA + B kb 9 7 5 3 1 Longer DNA fragments Shorter DNA fragments The observed electrophoretic pattern Restriction mapping: consider the possible arrangements: Which arrangements are correct? Possible maps of the 10-kb fragment: Enzyme A Enzyme B Treatment with restriction endonuclease A gave 2 fragments: one 7 kb in size and one 3 kb in size, as judged by gel electrophoresis. Treatment of another sample of the 10-kb DNA with restriction endonuclease B gave three fragments: 8.5 kb, 1.0 kb, and 0.5 kb. Treatment of a third sample with both restriction endonucleases A and B yielded fragments 6.5, 2, 1, and 0.5 kb. A B A + B 37 73 8.5 0.5 1 8.5 1 0.5 1 8.5 0.5 8.5 0.5 1 8.5 0.5 1 1 0.5 8.5 The only combinations giving the observed A + B digests are + and + 8.5 0.51 37 BB A Digests + 8.5 0.5 1 37 BB A Digests + To decide between these alternatives, a fixed point of reference, such as one of the ends of the fragment, must be identified or labeled. The task increases in complexity as DNA size, number of restriction sites, and/or number of restriction enzymes used increases. Treatment of a linear 10-kb DNA molecule with endonucleases gave the following results: FIGURE 10.29 Restriction mapping of a DNA molecule as determined by an analysis of the electrophoretic pat- tern obtained for different restriction endonuclease digests. (Keep in mind that a dsDNA molecule has a unique nucleotide sequence and therefore a definite polarity; thus, fragments from one end are distinctly dif- ferent from fragments derived from the other end.)