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 prote
Trang 1histone 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 A⬊U and G⬊C 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 3-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 coefficients1 (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 Mg2concentration is below 103M Each subunit is a
supramol-ecular assembly of proteins and RNA and has a total mass of 106D 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 S 10 13 sec.
Trang 2304 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 3-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'
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-dependentRNA 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 tocytoplasm
5' mRNA
snRNPs
Exon 1 Exon 2
3' AAAA
Translation mRNA is transcribed into a proteinby 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.
Trang 3carried to the ribosome attached as an acyl ester to the free 3-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 10 6 D) 70S
Subunits
(0.93 10 6 D) (1.59 10 6 D)
(1542 nucleotides)
23S RNA (2904 nucleotides) 5S RNA (120 nucleotides)
Protein
21 proteins 31 proteins
Ribosome
(4.22 10 6 D)
Subunits
(1.4 10 6 D) (2.82 10 6 D)
(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)
3
Pseudouridine ()
H
Dihydrouridine (D)
H H
H H N
N
N N
O H
N
O
Ribose
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.
Trang 4306 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 degradation 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 (Aminoimidazolecarboniaminoimidazolecarboni-trile 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.
C
H
OH C
H
H
H H
CH2OH
OH
OH HO
D -Ribose
C 5 H 10 O 5
C
C OH H
C
CH2OH
OH H
C OH H
Glycolaldehyde
C 2 H 4 O 2
-D -Glucopyranose
C 6 H 12 O 6
C
CH2OH
Adenine
N N
NH2
N
N H
Aminoimidazolecarbonitrile
H2N
C N
N
N H
䊱 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.
Trang 5found 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 C⬊G pair
to a U⬊A 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
2-OH group on each nucleotide in RNA is absent in DNA Consequently, the
ubiquitous 3-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 2-OH group makes the 3-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
2-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
2-OH; therefore, DNA is alkali stable
Cytosine
N
N
H O
NH2
+ H2O
Uracil
N
N
H O
H
+
NH3
O
FIGURE 10.25 Deamination of cytosine forms uracil.
O
CH3
N H O N
5 6
1 2 3
FIGURE 10.26 The 5-methyl group on thymine labels it
as a special kind of uracil.
Trang 6308 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 phosphodiesternucle-ases because they catalyze the cleavage of phosphodiester
O_
OH P O O O–
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–
OH P O O O–
OH P O O O–
U
OH
etc.
OH P O O O–
P O O O–
U OH P O O O–
G OH etc.
etc.
OH P O O O–
P O O
U OH P O O O–
G OH etc.
etc.
etc.
+
O O–
HO +
Sugar–PO4 backbone cleaved
O O
O
O O
H 2 O
H 2 O
or
C
OH
P O O–
O– 2'
3'
2'-PO 4 product
C OH 2'
3'
3'-PO 4 product
P O O–
O–
Complete hydrolysis of RNA by alkali yields a random mixture of 2'-NMPs and 3'-NMPs.
O
P O O O–
A
etc.
A
etc.
O OH
P O O O–
O
H 2 O
1
2
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 on the
P atom leads to 5 -phosphoester cleavage and random
hydrolysis of the cyclic 2 ,3-phosphodiester
intermedi-ate to give a mixture of 2 - and 3-nucleoside
mono-phosphate products See this figure animated at
www.cengage.com/login.
Trang 7bonds by H2O 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 3-side as a and the 5-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 3-end of the
poly-mer, whereas exo b cleavage involves attack at the 5-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
T
P
A
P OH
a b a b a b a b
Convention: The 3'-side of each phosphodiester
is termed a ; the 5'-side is termed b
Hydrolysis of the a bond yields 5'-PO4 products:
A
OH
G
OH
C
OH
T
OH
A
OH
Mixture of 5'-nucleoside monophosphates (NMPs)
Hydrolysis of the b bond yields 3'-PO4 products:
(a)
(b)
HO
C
HO
T
HO
A
HO OH
A 3',5'-diPO4
nucleotide
from the 5'-end
A mixture of 3'-NMPs
A nucleoside from the 3'-OH end
P
FIGURE 10.28 Cleavage in polynucleotide chains.
(a) Cleavage on the a side leaves the phosphate
attached to the 5 -position of the adjacent nucleotide,
while (b) b-side hydrolysis yields 3-phosphate products.
Trang 8310 Chapter 10 Nucleotides and Nucleic Acids
Restriction Enzymes Are Nucleases That Cleave Double-Stranded DNA Molecules
Restriction endonucleasesare 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 enzymeshave 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 5→3 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 5-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 5 -termini Other restriction enzymes, such as PstI,
which recognizes the sequence 5-CTGCAG-3 and cleaves between A and G, pro-duce cohesive staggered 3-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 5-TGGCCA-3 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
N N N N G A A T T C N N N N
N N N N C T T A A G N N N N
5 N N N N G A A T T C N N N N 3
3 N N N N C T T A A G N N N N 5
5 N N N N G A A T T C N N N N 3
3 N N N N C T T A A G N N N N 5
Trang 9About 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 5-PO4and
3-OH ends
Common Recognition
Enzyme Isoschizomers Sequence Compatible Cohesive Ends
Apy I AtuI, EcoRII CCgG(A)GG
BamHI GgGATCC Bcl I, Bgl II, MboI, Sau3A, XhoII
Bcl I TgGATCA BamHI, Bgl II, MboI, Sau3A, XhoII
Bgl II AgGATCT Bam HI, Bcl I, MboI, Sau3A, XhoII
EcoRII AtuI, ApyI gCC(A)GG
Hpa II CgCGG AccI, AcyI, AsuII, ClaI, TaqI
Mbo I Sau3A gGATC BamHI, Bcl I, Bgl II, XhoII
Sac I SstI GAGCTgC
Sau3A gGATC BamHI, Bcl I, Bgl II, MboI, XhoII
Taq I TgCGA AccI, AcyI, AsuII, ClaI, HpaII
Xho II (A)gGATC(T) Bam HI, Bcl I, Bgl II, MboI, Sau3A
TABLE 10.2 Restriction Endonucleases
Trang 10312 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 44nucleotides, 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 (46) 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
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
8.5
The only combinations giving the observed A+ B digests are + and +
1
A
Digests +
8.5
3 7
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.)