Polypeptide Polypeptide
DNA mRNA
A B
Ribosome
Ribosome
mRNA
Nucleus Cytoplasm
Transcription
Translation
Nuclear membrane DNA
(in chromosome) mRNA
Fig. 6.1 Expression of genetic information. In all organisms, the DNA of the gene is first copied into a single-stranded molecule of messenger RNA (mRNA). This process is called transcription. During ribosomal protein synthesis, the base sequence of the mRNA specifies the amino acid sequence of a polypeptide. This is called translation. A, In prokaryotic cells, translation starts before transcription is completed. B, Eukaryotic cells have a nuclear membrane. Therefore transcription and translation take place in different compartments: transcription in the nucleus, and translation in the cytoplasm.
67 DNA, RNA, and Protein Synthesis
5. Lysosomes are vesicles filled with hydrolytic en- zymes. They degrade cellular macromolecules and substances that the cell engulfs by endocytosis.
6. Peroxisomes contain enzymes that generate and de- stroy toxic hydrogen peroxide.
7. Cytoskeletal fibers give structural support to the cell.
They are also required for cell motility and intracellu- lar transport.
Differences between prokaryotes and eukaryotes are summarized in Fig. 6.2 and Table 6.1. Despite these dif- ferences, all living cells have three features in common:
1. They are surrounded by a plasma membrane, a flimsy, fluid, flexible structure that forms a diffusion barrier between the cell and its environment.
2. They generate metabolic energy, which they use for bio- synthesis, maintenance of cell structure, and cell motility.
3. They reproduce, transmitting their DNA through the generations and using RNA for gene expression.
DNA (chromosome)
Ribosomes
Plasma membrane Cell wall
MIT L L
L L L
L
L
L L
L
EV EV
EV EV
EV
SV
1 2 3 4 5 µm
SV
SV SV
SV G
R ER Cytoskeletal
fibers
P P P
P P
P
Nucleus
Nucleolu
s
A 1 µm
B
Fig. 6.2 Typical elements of prokaryotic and eukaryotic cell structure. A, Typical bacterial (prokaryotic) cell. B, Typical human (eukaryotic) cell. ER, Endoplasmic reticulum; EV, endocytotic vesicle; G, Golgi apparatus; L, lysosome; MIT, mitochondrion;
P, peroxisome; R, ribosome; SV, secretory vesicle.
Table 6.1 Typical Differences between Prokaryotic and Eukaryotic Cells
Property Prokaryotes Eukaryotes
Typical size 0.4–4 μm 5–50 μm
Nucleus – +
Membrane-bounded organelles
– +
Cytoskeleton – +
Endocytosis and exocytosis
– +
Cell wall + (some –) + (plants)
– (animals) No. of chromosomes 1 (+ plasmids) >1
Ploidy Haploid Haploid or diploid
Histones – +
Introns – +
Ribosomes 70S 80S
One difference is that in prokaryotes, transcription and translation occur in the same compartment. In eukary- otes, the two processes are separated: transcription in the nucleus, and translation in the cytoplasm.
DNA CONTAINS FOUR BASES
DNA is a polymer of nucleoside monophosphates (Fig. 6.3, B). Its structural backbone consists of alternat- ing phosphate and 2-deoxyribose residues that are held together by phosphodiester bonds involving carbon-3 and carbon-5 of the sugar. Carbon-1 forms a β-N-glycosidic bond with one of the four bases, as shown in Fig. 6.4.
One end of the DNA strand has a free hydroxyl group at C-5 of the last 2-deoxyribose. The other end has a free
hydroxyl group at C-3. The carbons of 2-deoxyribose are marked by a prime (′) to distinguish them from the carbon and nitrogen atoms of the bases; therefore, each strand has a 5′ end and a 3′ end. By convention, the 5′ terminus of a DNA (or RNA) strand is written at the left end and the 3′ terminus at the right end. Thus the tetranucleotide in Fig. 6.4 can be written as ACTG but not GTCA.
The variability of DNA structure is produced by its base sequence. With four different bases, there are 42 (or 16) different dinucleotides and 43 (or 64) different trinucleotides, and 4100 possibilities exist for a sequence of 100 nucleotides.
DNA FORMS A DOUBLE HELIX
Cellular DNA is double stranded, and almost all of it is present as a double helix, as first described by James Watson and Francis Crick in 1953. The most prominent features of the Watson-Crick double helix (Figs. 6.5 to 6.7) are as follows:
1. The two strands of the double helix have opposite polarity, meaning they run in opposite directions.
Base pairing is always antiparallel, not only in the DNA double helix but in all base-paired structures formed by DNA or RNA.
2. The 2-deoxyribose/phosphate backbones of the two strands form two ridges on the surface of the mole- cule. The phosphate groups are negatively charged.
3. The bases face inward to the helix axis, but their edges are exposed. They form the lining of two grooves that are framed by the ridges of the sugar-phosphate backbone. Because the N-glycosidic bonds are not exactly opposite each other (see Fig. 6.7), the two grooves are of unequal size. They are called the major groove and the minor groove.
4. In each of the two strands, successive bases lie flat one on top of the other, like a stack of pancakes.
The flat surfaces of the bases are hydrophobic, and successive bases in a strand form numerous van der Waals interactions.
5. Bases in opposite strands interact by hydrogen bonds. Adenine (A) always pairs with thymine (T) in the opposite strand, and guanine (G) with cytosine (C). Therefore the molar amount of adenine in the double strand always equals that of thymine, and the amount of guanine equals that of cytosine. A-T base pairs are held together by two hydrogen bonds, and G-C base pairs by three. Most important, the base sequence of one strand predicts exactly the base se- quence of the opposite strand. This is essential for DNA replication and DNA repair.
6. The double strand is wound into a right-handed he- lix. Each turn of the helix has about 10.4 base pairs and advances about 3.4 nm along the helix axis. The double helix is rather stiff, but it can be bent and twisted by DNA-binding proteins.
A
N H NH2
Adenine (A)
2 3 4
5
6 7
8 9
N1
N
N
N H H2N
Guanine (G) HN
N
N
Cytosine (C)
N H
2 1 6
5 4
N3
O
NH2
O
CH3
Thymine (T)
N H HN
O O
CH2
N
H H OH H
2-deoxy-AMP (dAMP)
B
NH2
N
N
N
O O–
O
–O P O
Fig. 6.3 The building blocks of DNA. A, Structures of the four bases, 2-deoxyribose, and phosphate. The bases A and G are purines, and C and T are pyrimidines. B, Structure of 2-deoxy-adenosine monophosphate (dAMP), one of the four 2-deoxyribonucleoside monophosphates (also called 2-deoxynucleotides) in the repeat structure of DNA.
Note that a nitrogen atom of the base is bound by a β-N- glycosidic bond to C-1 of 2-deoxyribose, whereas C-5 forms a phosphate ester bond.
69 DNA, RNA, and Protein Synthesis
DNA CAN BE DENATURED
Like other noncovalent structures, the Watson-Crick double helix disintegrates at high temperatures. Heat de- naturation of DNA is also called melting. Because A-T base pairs are held together by two hydrogen bonds and G-C base pairs by three, A-T–rich sections of the DNA unravel more easily than G-C–rich regions (Figs. 6.8 and 6.9). At physiological pH and ionic strength, this typically happens between 85 ° C and 95 ° C.
Heat denaturation decreases the viscosity of DNA solu- tions because the single strands are more flexible than the stiff, resilient double helix. It also increases the ultraviolet light absorbance at 260 nm, which is caused by the bases, because base pairing and base stacking are disrupted.
Other ways to denature DNA include decreased salt concentration, extreme pH, and chemicals that disrupt hydrogen bonding or base stacking.
When cooled slowly, denatured DNA “renatures”
spontaneously. This process is called annealing. Small
DNA molecules anneal almost instantaneously, but large molecules require seconds to minutes.
DNA IS SUPERCOILED
Many naturally occurring DNA molecules are circu- lar. When a linear duplex is partially unwound by one or several turns before it is linked into a circle, the number of base pairs per turn of the helix is greater than the usual 10.4. The torsional strain in this mol- ecule leads to supercoiling of the duplex around its own axis, much as a telephone cord twists around it- self. This is called a negative supertwist. The opposite situation, in which the helix is overwound, is called a positive supertwist.
Most cellular DNAs are negatively supertwisted, with 5% to 7% fewer right-handed turns than expected from the number of their base pairs. This underwound con- dition favors the unwinding of the double helix during DNA replication and transcription.
HO
5′-terminus N
N A
C
T
G NH2
N
N
N
O
N
O
NH2
N N
OH O
HN
N
N
O HN
O O O O
O
O O
O O
O CH3
H2N
3′-terminus CH2
–O P O
CH2
CH2
–O P O
CH2
–O P O
Fig. 6.4 Structure of the (2-deoxy-)tetranucleotide ACTG. The DNA strands in chromosomes are far larger, with lengths of many million nucleotide units. A, Adenine; C, cytosine; G, guanine; T, thymine.
The supertwisting of DNA is regulated by two types of topoisomerase. Type I topoisomerases cleave one strand of the double helix, creating a molecular swivel that relaxes supertwists passively. Type II topoisomerases are more complex. They cleave both strands and allow an intact helix to pass through this transient double- strand break, before resealing the break. Type II to- poisomerases relax positive supertwists passively and use ATP hydrolysis to pump negative supertwists into the DNA (Fig. 6.10).
DNA REPLICATION IS SEMICONSERVATIVE DNA makes identical copies of itself, which are trans- mitted to the daughter cells during mitosis and even to the next generation through the gametes. In this sense, DNA is the only immortal molecule in the body. The or- ganism is best understood as an artificial environment, created by genes for the benefit of their own continued existence.
DNA is replicated in two steps (Fig. 6.11):
1. The double helix unwinds to produce two single strands. This requires ATP-dependent enzymes to break the hydrogen bonds between bases. DNA un- winding creates the replication fork. This is the place where the new DNA is synthesized.
2. A new complementary strand is synthesized for each of the two old strands. This is possible because the
base sequence of each strand predicts the base se- quence of the complementary strand.
DNA replication is called semiconservative because one strand in the daughter molecule is always old and the other strand is newly synthesized.
DNA IS SYNTHESIZED BY DNA POLYMERASES Because a single-stranded DNA is required as a template for the synthesis of the new strand, unwinding of the double helix is required before the DNA can be rep- licated. A DNA polymerase then synthesizes the new DNA strand stepwise, nucleotide by nucleotide, in the 5′→ 3′ direction while reading the template in the 3′→ 5′ direction. The precursors are the deoxyribonucleoside triphosphates: deoxy-adenosine triphosphate (dATP), deoxy-guanosine triphosphate (dGTP), deoxy-cytidine triphosphate (dCTP), and deoxy-thymidine triphos- phate (dTTP).
DNA polymerase elongates DNA strands by linking the proximal phosphate of an incoming nucleotide to the 3′-hydroxyl group at the end of the growing strand (Fig. 6.12). The inorganic pyrophosphate that is formed
5′
3′
3′
5′
T A
G C
A T
C G
P
P
P
P
P
P P P P P
Fig. 6.5 Schematic view of the DNA double strand. Note that the strands are antiparallel and that only A-T and G-C base pairs are permitted. Therefore the base sequence of one strand predicts the base sequence of the opposite strand. A, Adenine; C, cytosine; G, guanine; P, phosphate; T, thymine.
Major groove
Minor groove
Sugar-phosphate backbone
Fig. 6.6 Space-filling model of the Watson-Crick double helix (B-DNA).
71 DNA, RNA, and Protein Synthesis
in this reaction is rapidly cleaved to inorganic phosphate by cellular pyrophosphatases.
DNA polymerases are literate enzymes. They read the base sequence of their template and make sure that each base that they add to the new strand pairs with the base in the template strand. Therefore the new strand is exactly complementary to the template strand. The DNA polymerases are lacking in creative spirit. They are like the scribe monks in medieval monasteries, who worked day and night copying old manuscripts without understanding their content.
DNA POLYMERASES HAVE EXONUCLEASE ACTIVITIES
The steps in DNA replication are best known in Escherichia coli, an intestinal bacterium that has en- joyed the unfaltering affection of generations of molec- ular biologists. The major enzyme of DNA replication in E. coli is DNA polymerase III (poly III), a very fast enzyme that polymerizes up to 1000 nucleotides per second. It also has very high processivity. This means it binds very tightly to the template strand. It does not fall
off the template until the entire bacterial chromosome has been replicated.
The major challenge in DNA replication is accuracy.
Nobody is perfect, and even DNA polymerase sometimes incorporates a wrong nucleotide in the new strand. The result is a mutation, a change in the nucleotide sequence that can be crippling or fatal if it leads to the synthesis of a faulty protein.
To minimize such mishaps, poly III is equipped with a 3′-exonuclease activity that it uses for proofread- ing. Nucleases are enzymes that cleave phosphodiester bonds in a nucleic acid. Deoxyribonucleases (DNases) cleave DNA, and ribonucleases (RNases) cleave RNA.
Nucleases that cleave internal phosphodiester bonds are called endonucleases, and those that remove nucleotides from the 5′ end or the 3′ end are called exonucleases.
The 3’-exonuclease activity of poly III comes into play only when the nucleotide that has been added to the 3′ end of a growing chain fails to pair with the base in the template strand. In this case the last nucleotide is removed (Fig. 6.13). This proofreading mechanism reduces the error rate from 1 in 104 or 1 in 105 to less than 1 in 107.
M i n
o r g r o o v e M
a j o
r g r o
o v e
N
N H H
N N
N N
CH3
Adenine
Thymine
N O
O
N
N N
N N H H
H H H
N N
Guanine
Cytosine
N
O O
H
O O
CH2
O 5′
3′
O CH2 O O
5′
3′
M i n o r g r o o v e M a j o r g r o o v e
O O
CH2
O 5′
3′
O CH2 O O
5′
3′
Fig. 6.7 Cross-sections through an adenine-thymine (A-T) and a guanine-cytosine (G-C) base pair in the DNA duplex. The A-T base pair is held together by two hydrogen bonds [–––] and the G-C base pair by three.
DNA synthesis is required not only for DNA repli- cation but also for DNA repair. DNA repair systems re- move damaged pieces from a strand and replace them by new DNA. The latter process requires DNA polymerases with low processivity. In E. coli, DNA polymerase I
(poly I) is the major repair polymerase, and three others (poly II, IV, and V) participate in specialized kinds of DNA repair.
Like poly III, many of the repair enzymes have proof- reading 3′-exonuclease functions. Some, including poly I, also have a 5′-exonuclease activity that participates in the removal of damaged DNA.
UNWINDING PROTEINS PRESENT A
SINGLE-STRANDED TEMPLATE TO THE DNA POLYMERASES
E. coli has a single circular chromosome with 4,639,675 base pairs and a length of 1.7 mm, almost 1000 times the length of the cell. The replication of this chromo- some starts at a single site, known as oriC. The 245 base-pair sequence of oriC binds multiple copies of an initiator protein that triggers the unwinding of the dou- ble helix. This creates two replication forks that move in opposite directions. Unwinding and DNA synthesis proceed bidirectionally from oriC until the two replica- tion forks meet at the opposite side of the chromosome (Fig. 6.14). The replication of the whole chromosome takes 30 to 40 minutes.
The first task, however, is strand separation, which requires an ATP-dependent helicase enzyme. The repli- cative helicase of E. coli is the dnaB protein.
Unwinding of the DNA causes overwinding of the double helix ahead of the moving replication fork. To prevent a standstill, positive supertwisting needs to be relieved by DNA gyrase, a type II topoisomerase.
DNA gyrase relaxes positive supertwists passively and induces negative supertwists by an ATP-dependent mechanism.
Once the strands have been separated, they are kept in the single-stranded state by associating loosely with a single-stranded DNA binding protein (SSB protein).
||
| | | | | | | || | |
Fig. 6.8 Melting of DNA.
70% G-C 30% G-C
50% G-C
Temperature (°C)
Relative absorbance
1.4 1.3 1.2 1.1
60 70 80 90
Fig. 6.9 Melting of DNA, monitored by the increase of ultraviolet light absorbance at 260 nm. The melting temperature increases with increased guanine-cytosine (G-C) content of the DNA. It is also affected by ionic strength and pH. The melting temperature is the temperature at which the increase in ultraviolet absorbance is half-maximal.
Positively supercoiled DNA
After relaxation of the supertwist, the ends of the DNA are linked by topoisomerase The supercoiled DNA is cleaved by topoisomerase, the free ends rotate to relax the supertwist
Fig. 6.10 Relaxation of positive supertwists in DNA by a type II topoisomerase.
73 DNA, RNA, and Protein Synthesis
+
Parental
duplex DNA
synthesis Replicated
daughter molecules Replication
fork
Movement of the replication fork
Fig. 6.11 Semiconservative mechanism of DNA replication.
CH2 O A T
P New strand
5′
CH2
CH2
P
5′
CH2
P 3′
P
O
OH
O
G
O
OH
T
P P P P P
P
O
OH C
C CH2
P
P P
2 Pi
O
A CH2
P O
G CH2
CH2 P
O
C CH2
P
Template strand O
CH2 O A T
P 5′
CH2
P
5′
CH2
P 3′
3′
3′
P
O
CH2
P
O
G
O
OH
T
C CH2
P O
A CH2
P O
G CH2
P O
C CH2
P O
Fig. 6.12 Template-directed synthesis of DNA by DNA polymerases. A, Adenine; C, cytosine; G, guanine; P, phosphate;
T, thymine.
ONE OF THE NEW DNA STRANDS IS SYNTHESIZED DISCONTINUOUSLY
None of the known DNA polymerases can assemble the first nucleotides of a new chain. This task is left to primase (dnaG protein), a specialized RNA polymerase that is tightly associated with the dnaB helicase in the replication fork. Primase synthesizes a small piece of RNA, 10 to 60 nucleotides long. This small RNA, base paired with the DNA template strand, is the primer for poly III (Fig. 6.15, A).
DNA polymerases synthesize only in the 5′→ 3′ di- rection, reading their template 3′→ 5′. Because the pa- rental double strand is antiparallel, only one of the new DNA chains, the leading strand, can be synthesized by a poly III molecule that travels with the replication fork.
The other strand, called the lagging strand, has to be synthesized piecemeal.
This requires the repeated action of the primase, fol- lowed by poly III. Together they produce DNA strands of about 1000 nucleotides, each with a little piece of RNA at the 5′ end. These strands are called Okazaki fragments.
The RNA primer is soon removed by the 5′- exonuclease activity of poly I. The gaps are filled by its polymerase activity, but poly I cannot connect the loose ends of two Okazaki fragments. This is the task of a DNA li- gase, which links the phosphorylated 5′ terminus of one fragment with the free 3′ terminus of another. Strangely, bacteria derive the energy for this reaction from the hy- drolysis of the phosphoanhydride bond in NAD, a coen- zyme that is otherwise used for hydrogen transfers (see Chapter 5), although humans use ATP (Fig. 6.16).
Fig. 6.15, B, shows that the enzymes of DNA repli- cation are aggregated in large complexes. The helicase is associated with the primase in a “primosome” to ensure that strand separation is followed immediately by syn- thesis of the new strand. DNA is synthesized by DNA polymerase III holoenzyme, a large complex with two
3′
3′ 5′
A
B
5′ 3′
Nuclease attack
3′ 5′
Nuclease attack 5′
Fig. 6.13 Exonuclease activities of bacterial DNA
polymerases. The products of these cleavages are nucleoside 5′-monophosphates. A, 3′-Exonuclease activity. Only mismatched bases are removed from the 3′ end of the newly synthesized DNA strand. This activity is required for proofreading. B, 5′-Exonuclease activity. Base-paired nucleotides are removed from the 5′ end. This activity erases the RNA primer during DNA replication and removes damaged portions of DNA during DNA repair.
t = 0 10 minutes
Replication fork
oriC Replication
fork
30 minutes 20 minutes
+
Fig. 6.14 Replication of the circular chromosome of Escherichia coli. Replication proceeds bidirectionally from a single replication origin (oriC). Dashed lines indicate new strands.
CLINICAL EXAMPLE 6.1: Gyrase Inhibitors Chemotherapeutic agents are weapons of mass
destruction that doctors use to exterminate undesirable life forms, such as bacteria, parasites, and cancer cells.
To be used effectively in the patient, chemotherapeutic agents must perform their mission without collateral damage to normal cells. As a rule, bacteria are more easily killed in the human body than are fungi and parasites because they are more different from human cells than are the eukaryotic pathogens. Cancer cells are most difficult to eradicate because they are too similar to the normal cells from which they evolved.
DNA replication is an attractive target because it is essential for the continued existence of all cells.
Ciprofloxacin and nalidixic acid are inhibitors of bacterial type II topoisomerases, including gyrase. They are used as antibiotics. Human topoisomerases are sufficiently different from the bacterial enzymes to be unaffected.
Drugs that inhibit human type II topoisomerases, including etoposide and doxorubicin, are used for cancer treatment. They have some selectivity for cancer cells because cancer cells divide more rapidly than normal cells and have more replication- associated topoisomerase action. These drugs do not prevent the initial DNA double-strand cleavage by the topoisomerase, but they delay or prevent the reconnection of the broken ends.