MCB 201 Gene Expression - Spring Semester 2003
Lecture 13 (Regulation of Transcription Initiation cont.)
1. As in prokaryotic cells, eukaryotic cells regulate gene expression most frequently at the level of initiation of transcription. Your text emphasizes how gene regulation in multicellular animals like mammals occurs most often as part of embryogenesis and cell differentiation pathways. In these situations, genes are often turned on or off permanently, i.e. until the cell dies. Single cell eukaryotes like yeast of course live more like bacteria, exposed to the environment and they are more responsive to environmental cues, as are bacteria. However, even mammals have conditions during which genes must be turned on and off relatively quickly, such as wound responses. Also, there are other environmental stress responses such as the heat shock and oxidative stress responses that are common to virtually all organisms.
2. Figure 10-22. How do we measure rates of transcription in animal cells? Here is outlined the experimental strategy behind a nascent-chain (run-on) assay for determining the rate of transcription of a gene. For this assay, nuclei are isolated from cells. Radioactively labeled NTPs are added for a five minute labeling period. The nucleotides are incorporated into nascent RNA transcripts by the RNA polymerases that initiated transcription before isolation of nuclei. Very few new initiation events occur in the isolated nuclei, presumeably because crucial components leak out of the isolated nuclei or are damaged during isolation. So only RNA chains started in vivo are elongated by 300-500 nucleotides, and thus the name run-on transcription for the assay. The nuclei are then lysed and the radioactive RNA is hybridized to a membrane filter to which cloned DNA for a specific gene has been attached. The complementary RNA will hybridize, the rest is washed away, and the filter is counted. The more radioactive RNA hybridized, the higher the rate of transcription. Relative rates of transcription can be measured in this way.
3. Figure 10-23. Experimental demonstration using run-on transcription of differential synthesis of 12 mRNAs encoding liver-specific proteins. Here we see that RNAs produced in nuclei isolated from liver cells (hepatocytes) hybridize to liver cDNAs but RNAs from kidney and brain do not. Some control DNA clones are also included such as cDNA encoding actin, alpha and beta tubulin, methionine tRNA and the plasmid DNA in which the cDNAs were cloned (cDNA is complementary DNA, produced in vitro by reverse transcriptase).
4. Figure 10-24. Construction and analysis of a 5'-deletion series to locate transcription-control sequences in DNA upstream of a eukaryotic gene. Regulatory elements in eukaryotic DNA are often many kilobases (thousands of bases) from start sites for transcription. These are regions where transcription factors bind. In this strategy, cloned DNA (carried in a plasmid vector) which includes the gene and regulatory region of interest is the starting material. The plasmid is cut with a restriction enzyme that cuts at one end of the cloned piece of DNA. Then an exonuclease is added and at increasing times, samples are taken and the exonuclease is inactivated. A piece of linker DNA carrying the restriction site for restriction enzyme C is attached using DNA ligase, the pieces are cut with restriction enzymes B and C, and DNA fragments created by deletions of increasing length are cloned into a plasmid vector in front of a gene encoding an easily assayed reporter enzyme. Note that the deletions are 5' to the predicted transcription start site and at least one deletion should remove the control elements and render this piece unable to direct the transcription of the reporter gene and thus the reporter enzyme will not be made. Each plasmid is tested by transfecting it into a population of cultured cells, allowing time for the reporter enzyme to be made, and then preparing a lysate of the cells for the enzyme assay.
5. Figure 10-25. The separation and identification of the three eukaryotic RNA polymerases by column chromatography. Eukaryotic cells have three different RNA polymerases. In this diagram, the separation of these molecules is shown using a salt gradient to differentially elute them from the column. Also, the individual fractions were assayed for their ability to carry out RNA synthesis in vitro from DNA templates and their sensitivity to alpha-aminitin, a highly toxic cyclic octapeptide from the Death Angel mushroom, which differentially inhibits these polymerases. It selectively inhibits Pol II at low concentrations whereas Pol III is sensitive to ten-fold higher concentrations at which Pol I still functions well. The three RNA polymerases transcribe different genes encoding different classes of RNA:
6. Figure 10-26. Schematic representation of the subunit structure of yeast nuclear RNA polymerases and comparison with E. coli RNA core polymerase. The core subunits called beta and beta-prime of E. coli RNA polymerase are similar in sequence to the two large (L,L') subunits of eukaryotic RNA polymerase and these are conserved in sequence among eukaryotes. These relationships suggest an ancient origin for this basic process of copying RNA using DNA templates. The earliest cells with DNA genomes probably had an enzyme recognizable as RNA polymerase, whose sequence has been substantially conserved during evolution.
7. Figure 10-27. Experimental demonstration that carboxyl-terminal domain (CTD) of RNA polymerase II is phosphorylated during in vivo transcription. For this experiment antibodies were raised in a rabbit against phosphorylated CTD and another antibody preparation was raised in a goat against unphosphorylated CTD. These antibodies were then incubated with salivary gland chromosomes from Drosophila. To determine where each form of CTD bound, antibodies that bind to the anti-CTD antibodies were incubated with the chromosome preparation (so-called second antibodies). These included anti-goat antibody labeled with fluorscein (green) and anti-rabbit antibody labeled with rhodamine (red). The result is that the puffed regions, representing regions of highly transcribed genes (activated by the molting hormone ecdysone), are stained red, indicating that they are sites of the phosphorylated form of CTD. CTD is the carboxyl-terminal repeat in the largest subunit of RNA polymerase II. This repeat is rich in serine and threonine residues which become phosphorylated when the polymerase binds to the promoter site and initiates transcription.
8. Color versions of figures in Gene Expression Minireview handed out in class: Figure 1, Figure 2, Figure 3, Figure 4.
