problem sets

Now to solve some problems:

Chapter 16: #1, 4-6, 8-15, 18, 19, 21, 22

16-1. a. induction, (4) stimulation of protein synthesis by a specific molecule

b. repressor, (8) negative regulator

c. operator, (5) site to which repressor binds

d. allostery, (2) protein undergoes a reversible conformational change

e. operon, (7) group of genes transcribed into one mRNA

f. catabolite repression, (1) glucose prevents expression of catabolic operons

g. reporter gene, (3) often fused to regulatory regions of genes whose expression is being monitored

h. attenuation, (6) gene regulation involving premature termination of transcription

16-4. Question: Would you expect mutations in the promoter that prevent binding of RNA polymerase to act in trans on another copy of the operon on a plasmid in the cell or in cis on the copy immediately adjacent to the mutated site?

Answer: I would expect it to act in cis because the promoter is the DNA sequence. Therefore, it cannot affect another cell

16-5. Question: Indicate whether the strain is inducible, constitutive, or unable to express beta-galactosidase and permease. Below are merodiploid strains containing lac operon alleles.

a. I+ O+ Z- Y+ / I+ Oc Z+ Y+

b. I+ O+ Z+ Y+ / I- Oc Z+ Y-

c. I+ O+ Z- Y+ / I- O+ Z+ Y-

d. I- P- O+ Z+ Y- / I+ P+ Oc Z- Y+

e. Is O+ Z+ Y+ / I- O+ Z+ Y-

Answer:

Permease Beta-galactosidase

a. constitutive constitutive

b. inducible constitutive

c. inducible inducible

d. constitutive no expression

e. no expression no expression

16-6. Question: Mutants were isolated in which the constitutive phenotype of a missense lacI mutation was suppressed. The operon was now inducible. These mapped to the operon but were not in the lacI gene. What could these mutations be?

Answer: The mutations could be the operator, which allows the missense mutation of the lacI gene to bind and thus repress the gene.

16-8. a. The DNA-binding protein is a negative regulator of gene expression because it suppresses transcription. Without this, the gene becomes constitutively expressed.

b. I should see that reg1 works in cis while reg2 works in trans because regulatory protein of reg2 is able to diffuse throughout the cytoplasm and act at target DNA sites on any DNA molecule in the cell. In contrast, the reg1 is a regulatory site that can influence only the expression of adjacent genes on the same DNA molecule that it is in. Reg2 should be dominant and makes all emu operon inducible, while Reg1 should affect only one operon, the operon that it is on.

I will work on the rest of the problems later. Now I must eat dinner.

Regulation of Gene Transcription

There are negative regulation and positive regulation. Negative refers to inhibition of RNA polymerase activity, while positive refers to enhancement of RNA polyemerase activity.

The gene for degrading the sugar lactose (found in milk) is comprised of lac permease and beta-galactosidase. The lac permease permits the lactose sugar to enter the cell, while the beta-galactosidase is necessary for cleaving the sugar bond. The lac gene is usually not active when the bacteria are in a medium that doesn’t have lactose. Adding lactose will induce the bacteria to express the lac gene.

The Operon Theory

The Operon Theory proposes to explain how repressors, promoters, inducers and other players work to achieve regulation of the gene for lactose utilization. The promoter (P) and the operator (O) are two regulatory elements that make up the lac operon: a single DNA unit enabling the simultaneous regulation of the three structure genes (lacZ, lacY, and lacA) in response to environmental changes.

In the negative regulation model, there is a repressor that binds to the operator and prevents RNA polymerase from binding to the promoter and begin transcription. However, an inducer in the form of lactose can bind to the repressor, inducing a conformational change that prevent the repressor from binding to the DNA. This then allows the RNA polymerase to bind to the promoter and begins transcription.

After the Operon Theory, a landmark in the field of genetics and biology, was proposed, people believed that negative regulation was the only regulative control available.

There was a key question, however, that brought up the idea of positive regulation. When bacteria grow in the presence of glucose and lactose, they do not express the lac gene. It is only when glucose is not available as an alternative source of fuel that bacteria start utilizing the lactose gene.

Positive control helps increase the transcription of lacZ, lacY, and lacA by enhancing the ability of RNA polymerase to bind and initiate transcription. CRP (catabolite activator protein), when bound to cAMP, enables CRP to bind to DNA in the regulatory region of the lac operon, increasing the ability of RNA polymerase to initiate transcription.

Glucose, however, regulates the level of cAMP by decreasing the availability of adenyl cyclase, which cleaves pyrophosphate from ATP to produce cAMP. It is only in the absence of glucose, therefore, that cAMP level rises.

Gene Regulation in Prokaryotes

This is Chapter 16 of Genetics—From Genes to Genomes, Second Edition; Hartwell, et al.

You might have suspected that since school season has already started, and is winding down for the holidays, that I will not have a lot of materials left to cover. You are right. Chapters 1 to 15 will be ignored. Let’s start!

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That chapter begins with an introduction to Vibrio cholerae, a bacterium that thrives in sewage and causes cholera. It describes how the bacteria are able to invade the digestive tract and release toxin that causes chloride ions to leak from the intestinal cells.

The goal of that introductory passage to prokaryotic gene regulation was to show that bacteria are able to respond to changes in the environment. In this case, when the V. cholerae bacteria enter through the mouth, they must handle the toxic acidity of the stomach, surviving until they reach the intestinal cells. After that, they must produce enzyme to degrade the protective mucus lining of the intestinal cells.

As usual, RNA Polymerase is the enzyme for transcription, the three steps of which include initiation, elongation, and termination. The sigma subunit is required for initiation. Two alpha, one beta, and one beta prime subunits make up the core enzyme. The sigma subunit recognizes the binds specific DNA sequence at the promoter.

After initiation, the sigma subunit is released to allow the core RNA polymerase to catalyze the polymerization of ribonucleotides, forming a complete mRNA.

Termination can be triggered with either of two signals: Rho-dependent, and Rho independent. Rho-dependent requires a protein factor called Rho to recognize a sequence in the newly transcribed mRNA. It then terminates transcription by binding to the RNA and pulling it away from the polyemerase enzyme.

In contrast, Rho-independent termination depends on a sequence of 20 bases in RNA that has a run of 6 or more uracils (U) at the end to form a stem loop, a secondary structure that serves as a signal for release of RNA polymerase.

Because unlike in eukaryotic organisms, prokaryotes do not enclose the DNA in a nucleus, translation of RNA can begin before transcription finishes. The ribosomes, the titular complex that link peptide bonds together by ways of tRNA to mRNA, start at special initation sites at 5′ end of the RNA reading frame.

Since this is basic Biology stuff, we’re really more focused on regulation. Regulation can occur during transcription, affecting the following actions: RNA polymerase binding to the promoter, shifting from initiation to elongation, or release of mRNA at the end of transcription. Post-transcriptional regulation can affect the stability of mRNA after synthesis (to ensure it doesn’t get over-translated), the stability of the polypeptide product, or the efficiency of ribosomes to recognize translational initiation sites.

To start with …

Yes, indeed, this blog is about studying. Since I love blogging, but I hate studying, I’m going to see if blogging helps me study. The format, hopefully, is this: I paraphrase what I read into the blog; I answer problems given from homework; and I may give up anecdotal studying tips. I reserve the right to change such format as necessary.

What am I studying? Molecular Cell Biology and Genetics are the two main courses I’m taking for my major. I’m also taking Computer Science and Chemical Ecology.

Why do I want to do this? I want to see if blogging is better than taking notes. I want to see if the act of publishing, of being visible on the Internet, will invigorates my interests, which have been flagging.