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1

Prokaryotic vs. eukaryotic gene organization

 

  • Define
    • Monocistronic
    • Polycistronic 

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

2

What is sigma factor?

  • 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. 

3

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

  • 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. 

4

What is restriction enzyme?

  • 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. 

5

Define:

  • Western Blot
  • Southern Blot 
  • Northern Blot

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.

6

What is the relevance of the SV40 virus?

It contained the first enhancer that scientists discovered

7

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

 

  • Type I restriction enzyme

    • cleaves some distance away from the recognition sequence

  • Type II restriction enzyme
    • cleaves within the recognition sequence

8

What is the difference between a negative or positive control?

  • 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.

9

What does promoter asymmetry mean?

  • 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

10

What is an insulator?

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

11

What does adenyl cyclase do?

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

12

How does cAMP interact with CAP?

  • 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. 

13

What exactly happens to mRNA before it is translated?

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

14

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

Insulators

15

 

 

 

How does an enhancer function?

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

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16

 

 

How do enhancers present a problem to transcription?

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

  • 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)

17

 

 

What is the CAT assay?

  • 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.[

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18

Why are insulators not position independent? 

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19

 

 

 

What does an enhancer bind?

  • 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...)

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