2) Briefly describe 6 different ways that eukaryotic cells typically regulate Gene Expression

1. Histone methylation can have many different effects on gene expression. In some cases, histone methylation is associated with activation of transcription, whereas in other cases it can trigger the formation of heterochromatin and a decrease in transcription. If histone methylation has been detected in the region of gene YFG in yeast, describe an experiment that could distinguish whether the methylation is important to activate or repress transcription of gene YFG. al 6 2. An E. coli strain of chromosomal genotype lacl* lacP" lacO* lacz* lacY*. You wish to transform this strain into a wild-type lac operon by the addition of an extra piece of DNA (plasmid). a. What regulatory region(s), gene or genes would you add on this extra DNA that would make this strain express the lac operon genes as wild type? b. How would you design this plasmid if you want the strain of E. coli to express the lac operon genes even if lactose is absent in the medium (constitutively)?

3. The average molecular weight of human proteins is about 50,000 daltons. A few proteins are much larger, such as a muscle cell protein called titin, which has a molecular weight of 3,000,000 daltons. B) If the nucleotides in the coding portion of the mRNA constitute 5% of the total nucleotides that are transcribed, how long will it take a muscle cell to transcribe a gene for an average protein versus the titin gene. Assume that the transcription rate is 50 nucleotides per second.

1. Which types of RNA are transcribed by each RNA polymerase enzyme in eukaryotic cells?

5. The following questions are about cis-regulatory elements (CRES). a. Describe the role of regulatory elements in eukaryotic gene expression b. What type of CREs are there? c. How do transcription factors 'know' where CREs are?

2. Shown below is a schematic of a prokaryotic gene. The light dotted area represents the region that is transcribed. represents the promoter, and the direction of transcription is shown by the arrow. The in m a. 3' 5' Which strand of the DNA is being used as a template for transcription, the top or the bottom? Why? b. On the diagram, indicate where the start codon would be. What sequence would it have? C. On the diagram, indicate where the stop codon would be. d. On the diagram, indicate where the transcription stop would be found.

7. Explain why a mutant Ras is an oncogene causing many human cancers

1. You are interested in the eukaryotic protein/enzyme Thiolase, which is 200 amino acids in length. Because Thiolase is only active in the peroxisome (an organelle) you know there must be a peroxisomal localization signal sequence in the protein. Sub-cellular Location Construct of GFP protein In order to be able to visualize the Thiolase protein in a cell, you create a fusion Cytosol construct with the gene for Thiolase and the GFP gene for Green Fluorescent Protein (GFP). 200 Thiolase Peroxisome GFP You also make a series of deletions in the 50 200 Peroxisome gene for Thiolase, all fused with the GFP GFP 100 200 gene. Numbers on top of the constructs GFP Peroxisome represent amino acid positions. 125 200 Cytosol GFP You express each construct in a mammalian 150 200 cell and use a microscope to visualize where the Thiolase-GFP protein gets trafficked. cytosol GEP 150 Peroxisome GFP Using the data provided, where must the peroxisomal localization signal sequence be in the Thiolase…

8. In genetic screens of mutagenized yeast, temperature sensitive mutants defective in the secretory pathway (sec mutants) were identified. These mutants have helped scientists study the protein secretion pathway that is necessary/essential for life in eukaryotic cells. To identify sec mutants: mutagenized yeast cells were grown for 12 hours at low temperature, shifted to higher temperature for two hours, and then the population of cells were centrifuged in a density gradient. a. Mutagenized cells were first grown at the lower temperature and then shifted to the higher temperature. i. Why was the shift in temperature an important step of the experimental protocol? ii. Why would the experiment have been unsuccessful if the cells were grown exclusively at the higher temperature? b. After centrifugation, where would sec mutant cells be located compared to other cells in the population? Briefly explain why.

6.  explain what coupled transcription-translation is, and why it occurs in prokaryotes but not eukaryotes