Tutorial

Molecular Cell Biology 7 Chapter 7: Transcriptional control of gene expression Richard Bartfai
1. Describe what happens at the molecular level with the lac operon when E.coli bacteria go from a glucose-containing medium to a lactose-containing medium. 2. The figure below describes the relation between the nucleotide sequence of the template and non-template strands of DNA and the corresponding RNA product. The author however has made three unfortunate mistakes in this illustration regarding transcriptional and translational initiation. Which are these mistakes?

3. You have identified a novel type of RNA with unknown function. Describe an experiment to determine if this RNA is transcribed in an RNA polymerase II dependent fashion. (There is more than one good answer!) 4. Searching for genetic alteration that occurs in all patients suffering from an inherited disease a mutation far away (>10kb) from any coding sequences is identified. What kind of regulatory element might be affected by the mutation? Please design an experiment to prove that this DNA element is indeed involved in gene regulation and the mutation disrupts this function. 5. Describe the protein domain structure of transcription activators and repressors. 6. Activator and repressor domains generally have little sequence conservation and are therefore difficult to be identified by bioinformatic means. Describe an experiment to identify the activation domain of a novel regulatory protein with a DNA-binding domain at its N terminus. (There is more than one good answer! Look for potential clues in Chapter 7) 7. Glucocorticoid, Thyroxine and Serotonin do influence gene expression of the target cell via different mechanisms? What are these differences? 8. Expression of recombinant proteins in yeast is an important tool for biotech companies in their effort to develop drugs and cures for human disease. In an attempt to express gene X in yeast, the gene was integrated in het yeast genome together with a selectable marker (for example an antibiotic resistance gene, so yeast cells can be selected that have the transgene integrated in their genome).

a. Do you expect to find yeast cells that have gene X and the selectable marker integrated close to one of the telomeres? Why? b. In a situation that you have a transgene integrated near a telomere, how will transgene expression be affected by deletion of the N-terminal tail of histone H3? Why? 9. Exam question a. Provide a short description and / or definition of the key terms listed below. Relate your description to the regulation of transcription if possible. i. 5 untranslated region (provide a description and comment on the ATG and the transcription start site) ii. histone modifications (provide an example of how these modifications influence transcription) iii. CTD phosphorylation b. Proteins involved in chromatin remodeling can, depending on the situation, activate or repress transcription. Explain how this is possible. c. Proteins that can stimulate transcription in a functional assay have been biochemically purified from a crude cell extract. A gel shift (also referred to as band shift or EMSA is performed with nucleosomal DNA in which a single nucleosome is present on a linear DNA fragment. The DNA is radioactively labeled and after incubation the samples are run on gel, the gel is dried, and radioactive DNA fragments are visualized on film. The mobility of the DNA-nucleosome complex in the non-denaturing gel depends on the total size of the complex and the position of the nucleosome on the DNA (see figure, schematic drawing of four different positions of the nucleosome on the DNA). Free DNA is also present in the mixture, but migrates very fast in the gel. In the experiment nucleosomal DNA is incubated in the presence or absence of the purified protein fraction, in the presence or absence of ATP. The result is shown below:

Fraction

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Guschin et al. (2000)

What are the properties of the proteins in the fraction regarding i. binding to DNA, ii. binding to nucleosomal DNA en iii. remodeling of nucleosomal DNA? In other words, what does the fraction contain? Explain why.