Lecture 1

Question 1

Prokaryotic vs. eukaryotic gene organization

• Define
  
  • Monocistronic
  • Polycistronic

Answer 1

Prokaryotic vs. eukaryotic gene organization

• Define
  
  • Monocistronic
    
    • A simple eukaryotic transcription unit produces a single monocistronic mRNA, which is translated into a single protein.
    • A complex eukaryotic transcription unit is transcribed into a primary transcript that can be processed into two or more different monocistronic mRNAs depending on the choice of splice sites or polyadenylation sites
  • Polycistronic
    
    • bacterial mRNAs are often polycistronic
    • Encode several different proteins, each of which is translated from the same mRNA molecule

Question 2

What is sigma factor?

Answer 2

• A sigma factor (σ factor) is a protein needed only for initiation of transcription.
• It is a bacterial transcription initiation factor that enables specific binding of RNA polymerase to gene promoters.

Question 3

• How does cAMP aid in the binding of RNA polymerase to the promoter?

Answer 3

• Binding of RNA polymerase to the promoter is aided by the cAMP-bound catabolite activator protein (CAP, also known as the cAMP receptor protein).
• CAP recruits sigma factor.
• cAMP binding to CAP allows CAP to bind to DNA, promoting transcription.

Question 4

What is restriction enzyme?

Answer 4

• A palindromic sequence of plasmid DNA that has a specific cut site that allows you to break DNA with either 3 prime or 5 prime overhangs.
• It allows DNA to be cut at specific sites.

Question 5

Define:

• Western Blot
• Southern Blot
• Northern Blot

Answer 5

Define:

• Western Blot
  
  • reveals a pattern of bands showing the size and relative amount of a particular protein.
• Southern Blot
  
  • reveals a pattern of bands showing the size and relative amount of DNA molecules containing the probe sequence.
• Northern Blot
  
  • reveals a pattern of bands showing the size and relative amount of RNA molecules containing the probe sequence.

Question 6

What is the relevance of the SV40 virus?

Answer 6

It contained the first enhancer that scientists discovered

Question 7

What is the difference between Type I and Type II restriction enzymes?

Answer 7

• Type I restriction enzyme
  
  • cleaves some distance away from the recognition sequence
• Type II restriction enzyme
  
  • cleaves within the recognition sequence

Question 8

What is the difference between a negative or positive control?

Answer 8

• A negative control is part of a well-designed scientific experiment.
  
  • A negative control group is a group in which no response is expected.
• A positive control is a control in which a known response is expected.

Question 9

What does promoter asymmetry mean?

Answer 9

• TATA box are sequences that help bind sigma factor so that the orientation tells polymerase which strand to replicate.
• Which end of the gene has the TATA box -> asymmetry.
• From the transcription start site
  
  • TATA BOX is minus 10
  • The second sequence is minus 30

Question 10

What is an insulator?

Answer 10

Insulators have two main functions

• It is part of the genomic sequence.
• Enhancer-blocking insulators prevent distal enhancers from acting on the promoter of neighboring genes
• Barrier insulators prevent silencing of euchromatin by the spread neighboring heterochromatin

Question 11

What does adenyl cyclase do?

Answer 11

• Convert ATP to cAMP
• It is allosterically regulated by glucose.
  
  • (+) low glucose
  • (-) high cAMP

Question 12

How does cAMP interact with CAP?

Answer 12

• cAMP binds to CAP to allosterically activate it
• CAP the binds to a site upstream of a promoter and recruits sigma factor and RNA polymerase.

Question 13

What exactly happens to mRNA before it is translated?

Answer 13

• Introns are spliced out
• Poly A tail added
• 5’ cap added

Question 14

What are DNA sequence elements that bind proteins with enhancer‐blocking and/or chromatin barrier activity?

Answer 14

Insulators

Question 15

How does an enhancer function?

Answer 15

• The enhancer appears to function to bring proteins into the vicinity of the promoter

Question 16

How do enhancers present a problem to transcription?

How is the action of enhancers restricted to prevent this problem?

Answer 16

• Enhancers are promiscuous
  
  • Enhancers located a great distance from their target genes have the potential to activate intervening non target genes
• Solution: Insulators!
• DNA sequence elements that bind proteins with enhancer‐blocking and/or chromatin barrier activity
• Insulator proteins have been extensively studied in flies (less so in humans)

Question 17

What is the CAT assay?

Answer 17

• CAT is used as a reporter system to measure the level of a promoter or its tissue-specific expression.
• The CAT assay involves monitoring acetylation of radioactively labeled chloramphenicol on a TLC plate; CAT activity is determined by looking for the acetylated forms of chloramphenicol, which have a significantly increased migration rate as compared to the unacetylated form.[

Question 18

Why are insulators not position independent?

Answer 18

Question 19

What does an enhancer bind?

Answer 19

• Transcriptional regulators! (General term: transcription factors)
• Lots of them, mostly conserved
• Have small DNA binding domains that recognize specific features of DNA
• Usually work in dimers (potential for combinatorial activity…)