MCB 201 Gene Expression - Spring Semester 2004

Lecture 3 (Structure of Nucleic Acids continued)

Topics:
 
1. Figure 4-6, Lodish4e and Figure 4-4, Lodish5e: Models of various DNA structures that are known to exist. This is a particularly useful figure for getting a clearer idea of the structure of the double helix since both side views and views looking top down along the helical axis are shown. a) The usual form of DNA in cells is the B form with a complete helical turn every 10 base pairs (3.4 nm). Note the major and minor grooves. b) The A form, found in RNA-DNA and RNA-RNA helices, is a more compact structure with 11 base pairs per turn, a large tilt of the base pairs relative to the central axis, and a central hole, seen in top view. c) Z DNA is a left-handed helix with a zig-zag appearance of the sugar-phosphate backbone, hence the name. d) A triple-helix can form when all purine bases in one strand are matched by all pyrimidines in the complementary strand, making room for a third polypyrimidine strand.
 
2. Figure 4-7, Lodish5e: Supercoiling shown in electron micrographs of DNA isolated from SV40 virus. Unlike most eukaryotic cell DNA, which is linear in the sense that is has free ends, all prokaryotic genomic DNAs and many viral DNAs are circular molecules. For example, when DNA extracted from the animal virus simian virus 40 is released from associated proteins, the DNA helix is underwound and has the supercoiled configuration shown as Form I. If one strand is nicked, the strands can rewind, producing the relaxed-circle configuration shown as Form II, which lacks supercoils. Topoisomerase I catalyzes this reaction and reseals the broken ends of the DNA strands. All of the supercoils in an isolated DNA molecule can be removed one at a time by the sequential activity of topoisomerase I. The diagrams under the electron micrographs show a simplified view of the supercoiled SV40 DNA double helix and the nicked, relaxed form.
 
3. Figure 4-7A and B, Lodish4e: The most important change in B form DNA structure, from the viewpoint of regulation of gene expression, comes from the binding of proteins to specific DNA sequences. This can result in both the bending of the axis of DNA and its untwisting to expose unpaired purine and pyrimidine bases. These changes have major functional significance for regulation of gene expression at the level of transcription because they open up the DNA for binding of additional transcription factors and help to make the DNA template available to polymerases to read the nitrogen bases in the DNA template.
 
Figure 4-5, Lodish5e: This is the space-filling model of a domain of the TBP protein bound to the TATA box in the minor groove of the DNA. This is an sequence of DNA rich in the bases A and T, which are each held together by two hydrogen bonds, compared to sequences rich in the bases G and C, which are each held together by three hydrogen bounds. The DNA double helix segment containing the more weakly associated A-T rich sequences is untwisted and bent sharply. This complex starts or initiates transcription of most eukaryotic genes.
 
 
4. Figure 4-8, Lodish4e: Denaturation and renaturation of double-stranded DNA. One of the most useful properties for the experimental analysis of DNA is its ability to undergo denaturation by heat and alkali conditions into single-stranded molecules and then to renature into a complementary based-paired double helix (nucleic acid hybridization). These processes occur naturally and transiently during DNA replication and can be produced experimentally in the lab.
 
Conditions that favor denaturation destabilize the DNA double helix:
 
a. Increased temperature.
b. Decreased ionic concentration of solution.
c. Addition of formamide or urea, both of which destabilize hydrogen bonding in the helix.
d. Alkaline or basic pH, which also destabilizes hydrogen bonding in the helix.
 
Conditions that favor renaturation stabilize the DNA double helix:
 
a. Decreased temperature.
b. Increased ionic concentration of solution.
 
5. Figure 4-6, Lodish5e: Light adsorption and temperature in DNA denaturation. What is the definition of Tm? How does Tm change with increasing GC content of DNA?
 
6. Figure 4-8, Lodish5e: RNA secondary structure and tertiary structure. The sugar-phosphate backbone of single-stranded RNA allows for a flexible structure that can fold into a variety of different three dimensional shapes. In this ability, RNA is more like proteins than it is like its chemically similar relative DNA. And in fact, some RNA called ribozymes have enzymatic activities as do proteins. The secondary structures like stem-loops and hairpins are stabilized in cells by proteins that recognize these regions in RNA as binding sites. These sites and their bound proteins are often involved in regulation of gene expression at the RNA level (translation).
 
7. "Meet the Scientists video" - Watson and Crick

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