基因的分子生物学英文版答案molecular chapter6

Chapter 6 Answers

1.

a. See Figure 6-6b

b. See Figure 6-6b

c. donors on adenine: exocyclic amino group at C6; N1

acceptors on thymine: carbonyl at C4; N3

2.

a. See Figure 6-6a

b. See Figure 6-6a

c. donors on guanine: exocyclic amino group at C2; N1

acceptors on guanine: carbonyl at C6

donors on cytosine: exocyclic amino group at C4

acceptors on cytosine: carbonyl at C2; N3

3. In addition to the energy contributed directly by the hydrogen bonds, and the decrease in entropy resulting from the displacement of water molecules from the bases in single-stranded DNA, the stacking interactions between the bases also contribute to the stability of the double helix. These involve electron cloud interactions between adjacent bases within the double helix.

4. Because DNA contains thymine, the cell can recognize uracil as a defective base resulting from the deamination of cytosine, and can repair it accordingly. If DNA naturally contained uracil, then it would be impossible to distinguish between the normal uracil bases in the DNA and the products of cytosine deamination, making repair impossible.

5. These relative proportions indicate that the X174 genome is single-stranded. If it were double-stranded, the proportion of A would be equivalent to that of T, and the proportion of C would be equivalent to that of G.

6. Because the major groove is larger than the minor groove, it is more accessible to the amino acid side chains of DNA-binding proteins. In addition, the major groove is richer in chemical information (more hydrogen bond donors, hydrogen bond acceptors,

nonpolar hydrogen atoms and methyl groups), and the information that is present is more useful for distinguishing between base pairs.

(A=hydrogen bond acceptor; D=hydrogen bond donor; H nonpolar hydrogen; M=methyl group)

5’-AATCGG-3’, major groove: ADAM/ADAM/MADA/HDAA/AADH/AADH

5’-AATCGG-3’, minor groove: AHA/AHA/AHA/ADA/ADA/ADA

5’-CCGATT-3’, major groove: HDAA/HDAA/AADH/ADAM/MADA/MADA

5’-CCGATT-3’, minor groove: ADA/ADA/ADA/AHA/AHA/AHA

5’-GGCTAA-3’, major groove: AADH/AADH/HDAA/MADA/ADAM/ADAM

5’-GGCTAA-3’, minor groove: ADA/ADA/ADA/AHA/AHA/AHA

7. DNA in cells deviates from the ideal B form by having increased overall pitch, with an average of approximately 10.5 base pairs per turn instead of 10 in the ideal B form. In addition, DNA in solution is irregular, including deviations at the level of the co-planarity of the base pairs (propeller twist) and in the precise degree of twist at each base pair, locally affecting the width of the major and minor grooves.

The B form of DNA, which is the most common form in cells, exhibits a right-handed helix with a pitch of 10 base pairs per turn. B form DNA has a wide major groove and narrow minor groove. A form DNA, which is also right-handed, is more compact, having 11 bases per turn. The major groove in A form DNA is narrower and deeper, and the minor groove is broader and shallower. The orientation of the base pairs is tilted in A form DNA relative to the orientation of the helix. Z form DNA is left-handed, and the glycosidic bond connecting the bases to the 1’ position of 2’-deoxyribose is either in the anti conformation (for pyrimidines) or in the syn conformation (for purines; this bond is always in the anti conformation in right-handed DNA). The alternation of the syn and anti conformations produces a zigzag effect, hence the designation “Z” DNA.

The degree of propeller twist and other deviations from the ideal B form DNA is largely determined by the identity of a given base pair and that of its neighbors. B form DNA is favored under conditions of high humidity, and A form DNA under conditions of low humidity. Z form DNA can occur within sequences containing alternating purine and pyrimidine residues, in the presence of high concentrations of positively-charged ions.

8. [insert figure #3463506044]

9. You could take advantage of the influence of DNA topology on the electrophoretic mobility of cccDNA, as described in Box 6.3. Any relaxed cccDNA having a length that is a precise multiple of the pitch of the DNA will migrate more slowly in an agarose gel than a relaxed cccDNA having a length that is not a multiple of the pitch. For example, if the pitch is 10.5, then relaxed cccDNAs having lengths of 3990 or 4011 (which, being multiples of 10.5, will have no writhe when fully relaxed) will migrate detectably more slowly than a cccDNA of 3995 (which, not being a multiple of 10.5, will have some writhe even when in its most-relaxed state). Therefore, to test the pitch of DNA in the presence of fictionium, you could make a series of cccDNA molecules of slightly different lengths, incubate them in the presence of topoisomerase I, and separate them in the presence of the novel element. Determination of the sizes of the slowest-running molecules will allow the calculation of the pitch of the DNA.

10. Because the DNA is not cut, the addition of ethidium will not affect the Lk, and so this will remain as 950. The addition of each molecule of ethidium will decrease the local twist of DNA by 26° (from about 10° to about 36°), resulting in one fewer turn of the DNA for every approximately 14 (360°/26°) bound molecules of ethidium. Therefore, if there are 110 bound molecules of ethidium, there would be a decrease of about 8 turns of the DNA (110/14). Accordingly, the addition of the ethidium will lower the Tw by 8. In the absence of any change of the Lk, this decrease in the Tw necessarily produces an equivalent increase in the Wr.

In this case, we know that the length of the cccDNA is 10,500 bases, and that the Lk is 950. Under physiological conditions, the pitch is about 10.5°, so here a simple assumption is that the Tw is 1000 (10,500/10.5), and the Wr = –50 (i.e., there are 50 negative supercoils). Accordingly, after the addition of the ethidium the Lk will be 950, the Tw will be 992, and the Wr will be –42.

11. a. twist, no writhe

b. twist and writhe

c. neither twist nor writhe

d. twist and writhe

e. twist and writhe

12. Topoisomerase I makes a single-stranded cut in DNA, allowing the uncut strand to pass through the cut strand in order to bring about linking number changes in increments of 1, and does not require energy. Topoisomerase II makes double-stranded breaks in DNA and allows a region of uncut double helix to pass through the cut, changing the linking number in increments of 2. Topoisomerase II requires ATP to function.

To specifically purify topoisomerase II, the simplest approach would be to rely on the requirement of this enzyme for ATP. For example, you could look for protein fractions that contained a cccDNA-relaxing activity only in the presence of ATP, and could specifically exclude fractions that were capable of relaxing cccDNA in the absence of ATP.

13.

a. Double-stranded DNA circles

b. Relaxed or negatively supercoiled DNA (with writhe equal to or greater than—in increments of 2—that of the starting DNA)

c. Relaxed or negatively supercoiled DNA (with writhe equal to or greater than that of the starting DNA)

14.At high pH, the 2’ hydroxyl of a ribose within an RNA chain can become deprotonated and attack the phosphate at the 3’ position of the ribose (similar to the reaction shown in Figure 6-33b, except at high pH the 2’ hydroxyl becomes deprotonated even in the absence of Mg2+), producing a 2’,3’ cyclic phosphate and a free 5’ hydroxyl.

a. At high pH, the 2’ hydroxyl of ribose can become deprotonated, and the resulting negatively-charged oxygen can attack the 3’ phosphate group, as shown in Figure 6-33

b. As DNA does not have a 2’ hydroxyl g roup, it cannot undergo this reaction.

b. At neutral pH, the Mg2+ bound to the hammerhead ribozyme deprotonates the 2’ hydroxyl of the ribose, thereby activating the hydroxyl group and allowing a nucleophilic attack on the scissile phosphate.

15. The protein shields the negative charges of the RNA moiety to allow it to bind effectively to its negatively-charged substrate.

Addition of small, positively-charged counter-ions, such as spermidine, can render the protein unnecessary, permitting enzyme activity even in the absence of the protein.

16. A bound Mg2+facilitates the deprotonation of the 2’ hydroxyl of the ribose within the nucleotide shown as N1, and the resulting negatively-charged oxygen attacks the phosphate group at the 3’ position of the ribo se. As a result, the RNA chain is cleaved at that position, giving rise to a 2’,3’-cyclic phosphate within the ribose of N1, and a 5’ hydroxyl within the ribose of the nucleotide shown as N2.

17.

a. A two-stranded structure converts the ribozyme into a true enzyme, as the cleavage reaction occurs within a separate molecule and thereby allows the ribozyme to act upon multiple substrates.

b. Single-stranded DNA can act as an enzyme in a manner similar to a two-stranded hammerhead ribozyme, that is by acting as a template for an RNA substrate that undergoes self-cleavage. The 2’ hydroxyl is only required in the RNA substrate, not in the DNA enzyme.

18. It is unlikely that proteins recognize RNA through the major groove, as is often the case with DNA, because RNA is often single-stranded or, if double-stranded, in the A form. Because the A form double helix has a narrow, not very easily accessible major groove, it is unsuitable for specific recognition by proteins.

Proteins may recognize specific RNA species by the overall structure of the folded RNA molecule, including specific structural features such as stems, loops, bulges, hairpins, and other distinct forms.

19. Relaxing a cccDNA (for example by incubating in the presence of topoisomerase I) can only remove all of the writhe in the DNA if the length of the DNA is an exact multiple of the pitch of the DNA. For example, for DNA having a pitch of 10, it is possible to eliminate all of the writhe in cccDNA of 1000, 1010, or 1020, base pairs, but not in cccDNAs of 1005, 1015, or 1025 base pairs (the writhe will always be at least 0.5). Because writhe affects the speed with which DNA moves through an agarose gel, these two types of cccDNAs can thus be distinguished using electrophoresis. Therefore, to determine the helical periodicity of the DNA, make a series of cccDNAs of slightly increasing length, and determine whether the

slowest migrating molecules are those having a length that is a multiple of 10, 11, or another value.