Bio151 WI22-23  Topic 5 Problem Set Answer Key

/in /by

Instructions:

  • Download this problem set, keep the questions in the document and please type your answers in a color other than black so that your answers are easily discernable. 
  • Complete this entire problem set – give robust, detailed responses – this is practice so use it as such.
  • If you get stuck or need clarification on any of these questions… just ask!
  • Upload this document with your answers to the appropriate TurnItIn link in our Moodle page in PDF format
  • Please be advised that TurnItIn is a plagiarism and similarity checking software
    • You are always welcome to work with other people and/or get help on the problem set, however…
    • Make sure that you close and put away all sources of information before writing your answers so that you can be sure that your answers are in your own words and to convince yourself that you understand the concept and can explain it yourself.
    • If your answers are too similar to either someone else’s responses (past or present) or from the internet, points will be deducted as the assumption will be made that the work you’re doing is not your own.

Complete the entire problem set.  For grading, a problem or two will be randomly chosen for grading of 10 points and will be graded for completeness and originality worth 5 points; 15 points total. 

Due date: Sunday, January  by 11:59pm

Question 1: General Questions About Transcriptional Regulation

A. Explain whether RNA polymerase is a sequence-specific DNA-binding protein or not, and why the answer to this question is important for the process of transcription.

B. Explain the basic process by which transcription is controlled by transcription factors: what are transcription factors, what do they do, and how do they control transcription (you can keep this explanation general).

Question 2: Positive and Negative Control of Transcription

Explain the difference between positive and negative control of transcription as described in the lectures and notes for this topic.

Question 3: The lac Operon Regulatory System

In this topic, we’ll use the lac operon in E. coli as an example of transcriptional regulation. 

For this regulatory system, explain whether or not the (i) sigma factor (sigma 70), the (ii) lac repressor and the (iii) CAP protein are negative regulators of transcription, positive regulators of transcription or neither.

*Note that sigma factor is not considered a regulatory transcription factor (it’s a general transcription factor), but think about what it does and whether it fits into either one of these two categories as defined in Question 2.

Question 4: lacI Mutants

Researchers have studied mutants in the lacI gene to understand the function of the Lac repressor protein and how it regulates transcription at the lac operon in E. coli. 

A. One of these types of mutants is a loss of function mutation in the Lac repressor protein (the protein is no longer functional).  This is called a lacI- mutant.  How would the lacI- mutant affect the function of the lac operon? 

B. How well would lacI- mutants do in an environment that has lactose as its sole sugar source? Would it be able to survive if lactose were the sole energy source in the cell?  Explain your reasoning.

C. One of those types of mutants is called LacIS mutants (“S” stands for super-repressor).  These repressor proteins continue to bind to the operator in the lac operon promoter, even in the presence of the inducer (i.e. lactose).  How would the lacIS mutant affect the function of the lac operon? 

D. How well would lacIS mutants do in an environment that has lactose as its sole sugar source? Would it be able to survive if lactose were the sole energy source in the cell?  Explain your reasoning.

E. What would happen if there were a loss of function in the sigma70 binding site in the lacI promoter?  What effect would this have on regulation of the lac operon?

Question 5: cAMP Regulation of the lac Operon

Describe how cAMP regulates the lac operon and why it makes sense that both lactose (directly) and glucose (indirectly) affect the transcription levels of this operon.

Question 6: lac operon Mutants

Researchers create mutations to understand how processes like transcriptional regulation work in the cell.  This is certainly true for the lac operon, which was one of the first transcriptional regulation systems studied in cells. 

You are given 6 mutants in E. coli, which carry one or more mutations in the lac operon or the lacI gene.

For each strain, indicate whether the lacZ gene product (B-gal) will be expressed in the presence of lactose and whether it will be expressed in the absence of lactose.  Explain your reasoning in each case.

  • A “plus” superscript indicates normal functioning part of operon or repressor gene
  • A “minus” superscript indicates loss of function
  • “P” region is where the RNA polymerase binds (sigma70 binding site)
  • “O” is the operator
  • “I” is the lacI gene
  • “Z” is the lacZ gene
  • “Oc” means “operator-constitutive” mutant which means that the operator is mutated such that the repressor can’t bind
  • “Is” means “super-repressor” which binds the operator, but doesn’t bind lactose (so never dissociates from the operator)

Part I: A-F

A. I+P+O+Z+ 

B. IsP+O+Z+ 

C. I+P+OcZ+

D. I–P+O+Z+ 

E. IsP+OcZ+ 

F. I+P–O+Z+ 

Part II: In which of these mutant strains would you see low constitutive levels of expression (not completely off, but not fully on – like a “3” on our scale from 0 to 10) in the absence of lactose and presence of glucose?

Question 7: Hypothetical Operon Transcriptional Regulation System

Now we are going to work on an example of transcriptional regulation at a different operon.  This chromosomal region is in bacteria and contains two genes, fadA and fadB, which are necessary for the breakdown of oleic acid, a fatty acid energy source.