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• Identify the mechanisms for gene expression control in the lac and trp operons

• Explain what transcription factors and regulatory proteins are and how they regulated gene expression in eukaryotes.

• Describe how chromatin structure (DNA methylation and histone modification) can affect gene expression

• Explain the process of protein degradation and its purpose

• Contrast the mechanisms regulating gene expression depending on when they act on: 1) regulating transcription, 2) posttranscriptionally (regulating the mRNA and protein synthesis) and posttranslationally (regulation of protein degradation)

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Jamar Ferry
Jamar FerryLv2
28 Sep 2019
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Related questions

1) In the wildtype (the DNA sequence most commonly found in nature) lac operon, transcription is severely down-regulated. A mutation in lacI that causes an early stop codon in the sequence (called a lacI - strain) results in an operon that has high levels of transcription of the polycistronic m-RNA. What type of regulatory protein is the LacI protein (hint: we covered this type of protein in lecture)?

The Lac regulatory protein is called a repressor. (is this correct)

2) If a bacterium with a wildtype lac operon is grown in the presence of lactose, high levels of transcription result for a while, however, eventually transcription is again down-regulated. If, on the other hand, the sugar IPTG is present (instead of lactose), transcription levels are high and remain high. What function do IPTG and lactose share? How are they differently affected by the operon product? (need help with this question)

Lactose and IPTG affected by the operon are both inducers of the Lac-Operon. (is this correct)

3) What is “epigenetics”? What forms of gene regulation are usually involved in epigenetic control of a trait? Why do we have such a limited understanding of epigenetic processes? (need help with this question)

Epigenetics is the study of phenotype changes that do not change the DNA sequence. Genes can be turned on and off based on environment situations of past generations, (parents or even grandparents) were exposed. Examples of some environmental factors could be diet, stress, smoking, drinking alcohol, and diseases. Histone modification and methylation are usually involved in epigenetic control of a trait. (is this correct)

QUESTION 1

How is a bone marrow transplant able to treat someone with leukemia?

The donor blood contains antibodies that destroy cancer cells

Donor blood contains stem cells that regenerate cells in the immune system

Bone marrow contains induced pluripotent stem cells that turn into red blood cells

Bone marrow contains tumor supressor genes that slow down leukemia growth

Bone marrow contains pluripotent human embryonic stem cells

1.2 points

QUESTION 2

What would happen if siRNAs designed to target mRNA from an oncogene were introduced into cancer cells?

Increased genomic instability would result from adding foreign nucleic acids

New mutations would arise in the oncogene

The oncogene would be converted back into a proto-oncogene

The siRNAs would compete with the oncogene's active site

Production of the oncogene protein would decrease

1.2 points

QUESTION 3

What is the purpose of chromatin-remodeling enzymes in human cells?

The enzymes control the rate of translation of an mRNA transcript

The enzymes add phosphate or methyl groups to histone tails

The enzymes slide nucleosomes farther apart or closer to each other

The enzymes send damaged proteins to the proteasome for degradation

The enzymes cause DNA looping to occur

1.2 points

QUESTION 4

If you wanted to resurrect the mammoth, how would you do it?

Use somatic cell nuclear transfer to insert the mammoth genome into the egg of an elephant

Use transcription factors to re-program adult cells from elephants into mammoth cells

Change the gene expression patterns of elephants to increase production of mammoth genes

Use siRNAs to decrease expression of elephant-specific genes in an elephant embryo

1.2 points

QUESTION 5

How is eukaryotic transcription initiated?

RNA polymerase is recruited to the start codon by transcription factors

RNA polymerase begins transcription at the enhancer sequence

RNA polymerase is activated by binding to GTP

An initiator tRNA binds to the ribosome

A complex of proteins forms at the promoter of a gene

The overall goal of the research project that we carried out in Experimental Biology is insertion of GFP into target lapA/EF-4 gene.

1. What is the overall goal of the research project that we carried out in Experimental Biology, and how does the experiment aimed at purification of your fusion protein fit in with the overall goal of our research? How does the subcellular localization experiment fit into the overall goal?

2. What method did we use to determine whether your fusion protein was a soluble protein or a membrane bound protein? What positive control did we perform? What result was obtained from the positive control? Was this expected? Explain.

3. Know the methods used for protein purification in general as described in class, and in the file; “protein purification.ppt”

4. What method did we use to attempt to purify your LepA/EF 4 – GFP fusion protein? Why did we use this method, what advantages are there compared to other protein purification methods?

5. What is “Protein A” and how did we use it?

6. What antibody did we use in our purification experiment and why did we use it? Why can’t we use the anti-EF 4/LepA antibody to purify your fusion protein?

DAY 18, NOVEMBER 7: INTRODUCTION TO IN VIVO FUNCTIONAL ASSAYS; ASSAY FOR EXPRESSION TOXICITY AT 30°C VS. 37°C

Purpose: Compare the growth of pPEM109, pIN III, and candidate plasmids in DH5alpha-F’lacIQ -/+ IPTG to compare toxicity of functional LepA/EF-4 (expressed from pPEM109) to empty vector control (pIN III), and EF-4-GFP fusion proteins.

Background: This assay is based on the observation that E. coli is extremely sensitive to elevated levels of LepA/EF-4 expression. In prior work it was demonstrated that LepA/EF-4 expression from pPEM109 is lethal to cells growing in the presence of IPTG [Yaskowiak and March, 1995). Furthermore, pPEM109 can only be maintained in strains that contain the mutated Lac repressor gene, lacIQ, which increases repression of the operon. The wild-type promoter of the lacI gene is rather weak, resulting in ~10 molecules of Lac repressor protein per wild-type cell. The lacIQ gene contains a “promoter-up” mutation that increases transcription and Lac repressor expression 10-fold, resulting in ~100 repressor protein molecules per wild-type cell (Müller-Hill et al., 1998). Strains that only have normal Lac repressor have a slightly elevated level of “leaky” expression from the lac promoter. It was shown by comparing two identical strains (one contained lacIQ and one contained lacI) that pPEM109 could only be maintained in the lacIQ containing strain.

If you consider the structure of LepA/EF-4, and its proposed function, why do you think it would be toxic if expressed at high levels levels? What kind of mutations in the lepA gene could give rise to a mutated protein that is not toxic when expressed? What do you predict for your LepA/EF-4 - GFP fusion protein, will it be toxic when expressed or not?

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DAY 19, NOVEMBER 9: BIOCHEMICAL ASSAY FOR SUBCELLULAR LOCALIZATION OF LEPA/EF-4-GFP (Lysis and Ultracentrifugation).

Background: LepA/EF-4 was originally found in the cytoplasm, membrane and periplasm when the protein was expressed from a plasmid [March and Inouye (1985) J. Biol. Chem. 260: 7206-7213]. The endogenous protein, expressed from the chromosomal gene in the absence of plasmid expression was located in the membrane [March and Inouye (1985) Proc Natl Acad Sci, USA Vol. 82: 7500-7504]. More recently it has been shown that the cellular location of LepA/EF-4 depends on the Mg2+ stress status of the cell [Pech et al. (2011) Proc Natl Acad Sci, USA Vol. 108: 3199-3203].

Here we will examine the comparative distribution of LepA/EF-4 and LepA/EF-4-GFP in cells by subcellular fractionation of native cellular protein extracts. Do you predict that your EF-4-GFP fusion protein will be localized to the cytoplasm, membrane, or both?

In contrast to our previous denaturing extract preparation, the native preparation maintains the overall shape/folding of proteins so they can localize appropriately prior to analysis. Looking ahead, how will the preparation be different in native vs. denaturing extracts?

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DAY 20, NOVEMBER 14: BIOCHEMICAL ASSAY FOR SUBCELLULAR LOCALIZATION OF LEPA/EF-4-GFP (SDS PAGE and Transfer)

Procedure:

SDS PAGE Analysis

1. Each section should have the following control samples to analyze from DAY 19:

Extract Fraction Treatment

pPEM109 Input +IPTG

pPEM109 Sol +IPTG

pPEM109 Mem +IPTG

In addition, each student should have 3 experimental samples for their candidate:

Extract Fraction Treatment

Candidate Input +IPTG

Candidate Sol +IPTG

Candidate Mem +IPTG

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DAY 21, NOVEMBER 16: BIOCHEMICAL ASSAY FOR SUBCELLULAR LOCALIZATION OF LEPA/EF-4-GFP (Western Blot detection)

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DAY 23, NOVEMBER 28: PURIFICATION OF NATIVE LEPA/EF 4 – GFP FUSION PROTEINS BY AFFINITY CHROMATOGRAPHY.

Purpose: In order to examine whether LepA/EF 4 – GFP fusion proteins retain function our prior experiments examined whether fusion proteins retained expression toxicity, and whether fusion proteins localized to the membrane. In order to examine other aspects of function, such as GTP – binding and ribosome - binding, purified protein is required. In this experiment we examine the feasibility of purifying LepA/EF 4 – GFP fusion proteins using affinity purification.

Background: Scientists are often interested in recovering proteins that have been produced in bacteria for research or therapeutic purposes. The process of protein purification from bacteria is medically relevant. It can be used as a method to mass-produce proteins of interest. For instance, the human insulin gene can be cloned into a plasmid and transformed into E. coli. The protein can then be purified and used to treat patients with diabetes.

The process of purifying a protein means separating this protein from other cellular components (proteins, DNA, ions, etc.). Protein purification is described in all biochemistry books, and in Chapter 5, Section A, of the textbook used in the Emmanuel College Biochemistry class (Fundamentals of Biochemistry, Voet, Voet and Pratt). This book is on reserve in the Emmanuel College Library. Here we will test the feasibility of using affinity chromatography, and magnetic beads to purify LepA/EF 4 fusion proteins in one step.

Preparation of magnetic beads for affinity purification of native LepA/EF 4. This procedure will be done the day before by the Emmanuel College Lab staff.

Background. The primary antibody that we employed to detect LepA/EF 4 – GFP in our Western blotting experiments can also be used to purify native, functional LepA/EF 4 – GFP fusion protein. Commercially available Protein A magnetic beads will be used for this purpose. Protein A is a very stable bacterial protein that has a high affinity for IgG produced from any animal species. Protein A magnetic beads contain tiny magnetized beads with Protein A covalently attached to their surface. Remember that the primary antibody that was employed in our prior experiments was affinity-purified rabbit IgG that was specific for GFP epitopes. The application of GFP primary antibody to Protein A magnetic beads will result in a tight, non-covalent attachment of GFP – specific IgG onto the magnetic beads. These beads would specifically recognize and bind to GFP that was present in a crude cell lysate. The application of a magnetic field could then be employed to collect the beads and the bound LepA/EF 4. By this method the collection and wash steps would be very rapid and very efficient. After washing away unbound proteins, purified LepA/EF 4 – GFP could be eluted from the magnetic beads. HOWEVER, the GFP IgG was non-covalently attached to the Protein A, and therefore some will leak out in the elution step. This would contaminate the purified LepA/EF 4 – GFP fusion protein with IgG. To avoid this problem, GFP IgG will be chemically cross-linked onto the Protein A magnetic beads by the procedure described in Sisson and Castor [Journal of Immunological Methods (1990), Vol. 127, 215-220].

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