Select any seven problems and provide careful, detailed, well-worked out answers. Problem sets should be word processed. I prefer that they be sent to me electronically, but dead trees are acceptable.
1. (a) As we described in class with a cartoon, single stranded RNA often forms hairpin turns that allow it to base pair with itself. Here is the sequence of one such strand, reading from the 5' end: CUAGAUGGUAGGUACGGUUAUGGGAUAACUCUG. Suggest how this strand might form a hairpin. That is, which bases would pair, and which would be in the turn? More than one answer is possible. A graphic would be the best way to answer this.
(b) The RNAFold server will make a prediction of the folding of any segment of RNA or single-stranded DNA. Submit the sequence above to this server. Compare your prediction to that of the server and comment on any differences.
2. (a) Some naturally occurring polynucleotide sequences are palindromic; that is, they are self-complementary about an axis of symmetry. Such a sequence is:
---TCAAGTCCATGGACTTGG----
---AGTTCAGGTACCTGAACC----
Such sequences can form a double hairpin, or cruciform, conformation. (Think of the shape if you push toward the center on each end of this sequence, and one strand bulges upward while the other bulges downward.) Show how this sequence might form such a conformation, showing clearly the symmetry and self-pairing.
(b) Restriction endonucleases are enzymes that cleave both strands of DNA duplexes at specific sites. The recognition sites for these enzymes often are palindromic.
Here are two palindromic DNA sequences:
5'-G-A-A-T-T-C-3'
3'-C-T-T-A-A-G-5'
5'-G-A-T-A-T-C-3'
3'-C-T-A-T-A-G-5'
The palindromes can be found by carefully placing a two-fold symmetry axis between the strands, perpendicular to the page.
(c) A crystal structure of EcoRV ocmplexed with its cognate DNA is available from the PDB (code 1sx8). Read the paper reporting this structure [Biochemistry, 2004, 43, 6841] and examine the structure in Jmol. Try to determine what specific interactions enable the enzyme to recognize its binding site.
Use BioEdit (or other software of your choice) to translate it into an amino acid sequence. Tabulate (graphically) the frequencies of occurrence of the bases in the DNA and of the amino acids in the protein. (A tutorial for BioEdit is HERE.)
4. One result of Chargaff's demonstration that A+G = T+C was to disprove the suggestion that DNA was a monotonous tetranucleotide polymer; that is, that it was simply (ATCG)n. What experiments did he do to arrive at this conclusion? How does his demonstration of the cited ratio disprove the tetranucleotide hypothesis?
A second set of Chargaff's experiments involved degrading a sample of DNA with a deoxyribonuclease enzyme for different lengths of time, and then examining the base composition of the remaining undegraded DNA. Here are some data:
| Base | Intact DNA | 19% Left | 8% Left |
|---|---|---|---|
| Adenine | 0.27 | 0.33 | 0.35 |
| Guanine | 0.22 | 0.20 | 0.20 |
| Cytosine | 0.22 | 0.16 | 0.14 |
| Thymine | 0.27 | 0.26 | 0.23 |
How do these data further demolish the tetranucleotide hypothesis?
5. Consult the tabulation of the genetic code in the text or on our web site, and the web page showing structures and properties of amino acids. Answer these questions:
(a) Provide the structures of the amino acids coded for by codons in which the first two letters are the same. What are the codons?
(b) What similarities do you see in the structures of the amino acids identified in (a)?
(c) Several amino acids coded for by multiple codons can have any one of the four bases as the third letter. Which ones are they?
(d) The examples in (a) and (c) are the reason why Crick called the third base of a codon the "wobble" base, and suggested that the third base does do bind as tightly to its opposite in the anticodon of tRNA . Specifically, what advantage could there be to an organism in having the third base bind weakly?
6. Write a "curly arrow" mechanism for the cleavage and reconnection of a strand of DNA by topoisomerase IA.
7. In 1961, Sydney Brenner and Francis Crick definitively demonstrated that the DNA code uses triplets. They did this making use of some clever chemistry that allowed them to insert or delete single base pairs into a strand of DNA. What was the chemistry? Explain how this technique could be used to demonstrate the Code is a triplet code.
8. Over the period 1961-1965 Marshall Nirenberg and Gobind Khorana and their groups learned to synthesize chemically pieces of mRNA consisting of single bases, for example, AAAAAAAA....., or simple repeating patterns, like ACACACACACAC... This technique enabled them to crack the code, and win the Nobel prize for physiology or medicine in 1968. Explain how those pieces of RNA could be used to establish the code.
9. Many scientists believe that life on earth started in an "RNA World". That is, RNA preceded both DNA and proteins as the genetic medium and the catalytic medium for life processes. In this view, the large amount of noncoding or functional RNA produced from our DNA is a relic of this early world. Read the paper by Joyce, Nature, 2002, 418, 214 and discuss some of the evidence for RNA world and how the transition to DNA world might have come about.
10.Gene duplication has been recognized as an important part of evolution since the 1930s. Read these papers: Zhang, Trends Ecology Evol., 2003, 18, 292; Hurles, PLoS Biology, 2004, 2, 0900. Discuss how gene duplication occurs and what can happen to a duplicated gene.
11. A phenomenon called "gene silencing" has been discovered recently. It involves small interfering RNAs binding to mRNA and tagging it for destruction before it can be translated into protein. Provide a careful, complete description of how this genetic control works. Starting points: Nature, 2002, 418, 244; Nature, 2004, 430, 161; Nature, 2004, 431, 338; Science, 2002, 296, 1260ff.
12. Do problem #13 at the end of Chapter 28 in Lehninger (5th ed.). Include with your answers pictures to help illustrate them. The web page explains how to capture images.
For those working with the 4th edition:
To examine the roles of hydrogen bonds and hydrophobic interactions between transcription factors and DNA, go to FirstGlance: http://firstglance.jmol.org. Enter the PDB id 1tgh in the query box.
This file contains the crystal structure of a human TATA-binding protein and a segment of double stranded DNA.
Provide appropriate text and pictures to describe clearly how the molecules interact.