MCB 201 Gene Expression - Spring Semester 2006
Lecture 8 (The 3 Roles of RNA in Protein Synthesis cont., and DNA Replication)
8. Figure 4-30, lodish5e: Eukaryotic mRNA forms a circular structure owing to interactions of three proteins. This is a force-field micrograph showing circular mRNA that forms by the binding of purified poly(A)-binding protein I and the eukaryotic initiation factors eIF4E and eIF4G. The 5' and 3' ends of the mRNA in these structures is held together by protein-protein and protein-mRNA interactions. This is shown in the diagram on the next slide.
9. Figure 4-31, Lodish5e: Formation of circular eukaryotic mRNA by protein-protein interactions bridging the 5' and 3' ends. Force-field electron microscopy has been used to demonstrate the ability of eukaryotic mRNA to form circles. There is experimental evidence that these circles are held together with proteins. The poly(A)binding protein (PABI) at the 3' end of the mRNA interacts with a complex of proteins called eIF4 at the 5' cap of mRNA. Model of protein synthesis on circular polysomes and recycling of ribosomal subunits. What is the functional value of forming mRNA circles. One hypothesis is shown here. When the release step occurs in translation, the ribosomes dissociate into small and large subunits which along with initiation factors are reused by the cell to make another initiation complex. Since this complex usually forms on the 5' end of the mRNA, having the 3' end with the stop site nearby would keep the released components in the same vicinity as the new start site from the next round of translation.
Media Connections: Overview Animation: Life Cycle of an mRNA.
Section 4.6 (DNA Replication)
Introduction: Although the concept of copying a DNA molecule is relatively straightforward, the enzymology required for this process in the cell is actually quite complex. As it turns out, a large number of proteins and enzymes are required for DNA replication. Many proteins are required for replication because DNA polymerases, which add nucleotides to the 3' end of a DNA molecule, cannot perform a number of other steps required for replication (listed below).
A. DNA polymerases cannot melt double stranded DNA (dsDNA).
B. DNA polymerases cannot initiate DNA synthesis.
C. DNA polymerases cannot extend a DNA molecule from the 5' end.
To accommodate the inability of DNA polymerase to perform the above three functions, the cell requires enzymes to melt the DNA and initiate DNA synthesis. In addition, a rather awkward mechanism is employed by the replication machinery to accommodate the inability of polymerase to extend a 5' end of a DNA molecule. In this lecture we will discuss these reactions in more detail.
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1. There are three general features of DNA replication that must be accommodated in models of the replication process in all organisms:
A. Semiconservative replication
B. Bidirectional growth of new strands
C. Growth of new strands starting from a common site (replication origin)
2. The model of DNA proposed by Watson and Crick in 1952 suggested to them that a template model for DNA replication using regular rules of base-pairing (Chargaff's rules). This model could work by either a conservative or a semiconservative mechanism. The term conservative comes from the idea that the two parental DNA strands remain together and two new daughter strands are synthesized. In a semiconservative mechanism, the parental strands would come apart like a zipper being unzipped as the base-pairing interactions were broken. The phosphodiester bonds of the DNA strands would not break. Daughter strands would be synthesized, resulting in two double-stranded molecules in which one strand is parental and the other strand is a new daughter strand.
3. Figure 4-32. Matthew Meselson and Frank Stahl performed a classic experiment in 1958 showing that a semiconservative mechanism is used. The Meselson-Stahl experiment has been called the most beautiful experiment in science. While beauty is in the eye of the beholder, this was the perfect experiment for testing this hypothesis. This experiment is explained in the legend to this figure in a fair amount of detail. Here I would like to emphasize several things that made this experiment possible. First was the availability of a heavy mass isotope of nitrogen, 15N, which was produced in nuclear reactors. Ammonium salts of heavy nitrogen, as it was called, could be fed to the bacterium E. coli in culture medium. The heavy nitrogen was incorporated into newly made cellular molecules that contained nitrogen, including the nitrogen bases of DNA. In this way, it was possible to label both strands of the DNA double helix, by growing E. coli through multiple rounds of cell division in the heavy nitrogen medium. Then the cells were transferred to growth medium containing normal 14N, or light nitrogen. Predictions could be made about the labeling patterns of newly made DNA strands as shown in Panel A. The predictions were different depending on a conservative or a semiconservative mechanism of replication. The number of generations of cell division also affected the predictions. Cells were isolated after increasing generations, the DNA extracted, and banded according to mass in a density gradient. This method was possible due to advances in a method called eqiulibrium density ultracentrifugation in gradients made of cesium chloride. Some of these methods were developed by Dr. David Yphantis who later came to the University of Connecticut. UCONN is still home to the National Analytical Ultracentrifugation Center, now directed by Dr. James Cole. In Panel B, notice how the density patterns change with increasing numbers of cell generations (replication events). The key is the prediction and finding of an intermediate density double helical DNA molecule with a parental heavy strand and a daughter light strand (H-L). This intermediate is predicted to occur for semi-conservative replication but would not be seen during conservative replication.
4. Primer-template diagram. DNA polymerases require a primer to initiate replication. This primer may be either DNA or RNA. The primer is base-paired to the template strand of DNA, and a DNA polymerase adds deoxynucleotides to the free -OH group at the 3' end of the primer. Each base is chosen according to Chargaff's rules of complementary base pairing. When RNA is the primer, as when strans start up during replication, the daughter strand that is formed is RNA at the 5' end and DNA at the 3' end.
Media Connection: Nucleotide polymerization.
5. Figure 4-33. Schematic diagram of leading-strand and lagging strand
DNA synthesis at a replication fork. Here we put this primer-template diagram
into the context of an active replication fork. Each new daughter strand
grows in the 5' to 3' direction, indicated by the green arrowheads. This
requirement of a single direction forces synthesis on the leading and lagging
strands to proceed differently. On the leading strand, the daughter DNA
strand is synthesized continuously starting from a single RNA primer. The
lagging daughter strand is synthesized discontinuously using many RNA primers.
Space for an RNA primer is created each time the replication fork moves
due to unwinding of the parental DNA duplex. The DNA elongated on these
RNA primers occur in small fragments called Okazaki fragments after their
discoverer. As each Okazaki fragment moves up to a primer, the primer is
removed, DNA continues to fill in, and eventually the DNA pieces are ligated,
i.e. the final phosphodiester bond between them is made.
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