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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


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. 


  • 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. 


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. 



  • Western Blot
  • Southern Blot 
  • Northern Blot


  • 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.


What is the relevance of the SV40 virus?

It contained the first enhancer that scientists discovered


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


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.


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


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


What does adenyl cyclase do?

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


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. 


What exactly happens to mRNA before it is translated?

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


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






How does an enhancer function?

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

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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)




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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Why are insulators not position independent? 

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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...)