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MCB 110 Problem set 1. Nucleic acid structure – Answers

1. Imagine that you’re walking in the minor groove of double-stranded (ds), B-form DNA. Your hands are running along the phosphodiester backbone as banisters. a. Would there be a unique polarity to the phosphodiester bonds running through your hands? Yes. b. If so, what is the polarity for each hand? L hand: 5’-3’; R hand 3’-5’ c. Does this polarity change when you turn around and walk the other way? No! The strands are anti-parallel. d. Take a walk through the major groove and answer the same questions. R hand: 5’-3’; L hand 3’-5’ e. If you’re walking in the major groove and your classmate is walking toward you in the minor groove, will you ever meet? No, we’d face each other across the base pairs. f. As you walk in the grooves, you encounter three kinds of groups on the bases just as proteins do: H-bond donors, H-bond acceptors and bulky hydrophobic groups. Write a list of these groups and label them according to the scheme below.

For example: T, C5-methyl, hydrophobic, major groove = the methyl group on the C5 position of thymine. It sticks into the major grove. Major groove. T - C5 methyl, hydrophobic; C4 exocyclic keto: H-bond acceptor A - N7: H-bond acceptor, C6 exocyclic amino: H-bond donor G - C6 exocyclic keto: H-bond acceptor; N7: H-bond acceptor C – C4 exocyclic amino: H-bond donor Minor groove T - C2 exocyclic keto: H-bond acceptor A - N3: H-bond acceptor G - C2 exocyclic amino: H-bond donor; N3: H-bond acceptor C - C2 exocyclic keto: H-bond acceptor g. Do you ever encounter the same group in both grooves? No.

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2. List two things wrong with this figure of B-DNA. 3’ 5’

Minor groove Major groove

Bp spacing is 3.6 Å (not 3.6 nm), and the arrows on the backbones point the wrong ways.

5’ 3’ 3. Below is a space filling model of two nucleotides in a self-splicing RNA. Draw a stick representation of these nucleotides and label each atom and any implied hydrogen bonds between the bases.

It’s a symmetric A-A pair. The exocyclic amino group on position 6 of each base donates a hydrogen bond to the N1 nitrogen. 4. The persistence length of dsDNA (the length in which the chain turns around on average in solution) has been measured at 480-495 Å. a. How many base pairs does this correspond to in B DNA? X Å/(3.4 Å/bp) = 141-146 bp b. What makes the direction of the dsDNA “turn around” in solution? Mostly thermal fluctuaions. c. Does this make dsDNA stiffer or floppier than unstructured polypeptides and oligosaccharides? Much stiffer. Polypeptides turn around in ~3-4 amino acids. This corresponds to ~12 Å. d. When dsDNA is denatured, it absorbs ~40% more light at 260 nm. The hypochromism of dsDNA is thought to arise from the electronic interactions between the stacked bases. Denatured DNA absorbs approximately the same amount of light at 260 nm as a solution of mononucleotides with the same base composition. What do you conclude about the extent of base stacking in ssDNA? Is ssDNA likely to be stiffer or floppier than dsDNA? On average, ssDNA is unstacked, like free nucleotides. Therefore, ssDNA is floppier that dsDNA.

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5. The GC contents of different genomes are not correlated with the preferred growth temperatures of the various organisms. How is the stability of DNA controlled in vivo? In vivo, dsDNA forms closed loops (domains) or circular genomes (in bacteria). The tendency of the dsDNA to form ssDNA loops is controlled by supercoiling. R-handed supercoiling (overwinding) makes it harder to denature the dsDNA, while L-handed supercoiling makes it easier to open the duplex. Helicases and ssDNA binding proteins favor the open state. dsDNA binding proteins favor the duplex. 6. List at least three reasons that RNA adopts many more kinds of structures than DNA that has the same sequence. 1. 2’-OH H-bonds. E.g. ribose zipper or base contacts. 2. No complementary strand. 3. Metal binding sites involving 2’-OHs. 7. List three kinds of evidence for secondary structures (stems, loops, bulges) in RNA. In other words, how would you discover which regions of a specific RNA form secondary structures? E.g. Crystal structures, cleavage with ss-specific or ds-specific nucleases, base pairs conserved in homologous RNAs from different species, chemicals rapidly modify ss regions.