Lecture 1 Flashcards Preview

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

• 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. 

• 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. 

• 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. 

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.

It contained the first enhancer that scientists discovered

• Type I restriction enzyme

  • cleaves some distance away from the recognition sequence

• Type II restriction enzyme

  • cleaves within the recognition sequence

• 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.

• 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

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

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

• 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. 

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

Insulators

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

• 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)