Chapter 8-9: Nucleotides and Nucleic Acids Most of the material that you will be tested on comes from Chapter 8. Chapter 9 is great reading for enrichment, since of you have encountered techniques like CRISPR/Cas9 and molecular cloning in other classes and your labs. However, you will not be explicitly tested on how these technologies work. This was divided into two lectures. The first covered the basics of nucleotide structure, the structure of DNA, and changes that can occur to DNA. The second focused on three-dimensional structures of RNA, DNA, and binding proteins. Lecture material (part I): Memorize the structures of A, T, C, G, and U as the base by itself, the nucleotide, the nucleoside, and the 2’-deoxy version. You should be able to recognize these quickly. See figure 8.4, where they are all in one place. Which of these are purines and which are pyrimidines? You do not need to know the structures of minor bases. You must also memorize how “canonical” base pairs form. These are the classic Watson-Crick A-T and C-G pairs. Can you draw the H-bonds? Which of the two pairs is stronger? You should understand how the DNA double helix is formed: 3’-5’ phosphodiester bonds forming a sugar-phosphate backbone, Watson-Crick base pairing, phosphates on the outside, bases paired in the middle, all of which form a major and a minor groove. What forces hold the duplex together? How do H-bonding, solvent effects, and base stacking contribute? How do the A-form and Z-form of DNA differ from the common biological B-form? You should be able to identify major mechanisms of non-enzymatic DNA degradation, depurination and deamination. What causes these events? Sometimes it’s chemical, and sometimes it’s spontaneous. You should understand that these nonenzymatic changes happen very slowly and very rarely on a per-base basis, BUT that, summed over an organism’s entire genome and lifespan, mutations like these happen fairly frequently (and this is why we have extensive DNA repair systems). Why is it so important that DNA fidelity remains high, compared to other biomolecules, like proteins? Nucleic acid chemistry is also very important. How do we denature DNA? Which denatures at a lower temperature, a C-G rich region of DNA, or an A-T rich region? You should understand how basic, gel-based Sanger sequencing works. ! Lecture material (part II): You should understand secondary structure in DNA or RNA. You should be able to identify palindromes (these are not secondary structures; they are sequences that tend to form secondary structure). You should be able to identify secondary structuresincluding hairpins and cruciforms, bulges, internal loops, and guanosine tetraplexes. What are Hoogsteen positions? You should be able to think of a few examples of structural RNA molecules, such as tRNA, riboswitches, ribozymes, ribosome. You should understand that secondary structures fold into more complicated three-dimensional tertiary structures that we can only understand using structural techniques like NMR and x-ray crystallography. And finally you should understand how proteins interact with nucleic acids. We had several examples that illustrated a few common properties. DNA binding proteins have lots of positive charge (what specific sidechains?), and an a-helix can fit perfectly into the major groove. What is special about protein dimers in DNA binding? What is special about palindromic sequences? (We had two examples – lamba repressor and Cas9. Palindromes were important to both, but for different reasons.) Be able to describe the importance of the Rossman fold, and properties of this fold that enable nucleotide binding.