Meeting 13

  1. control of Tx initiation:
    is the most important step for determining whether genes are expressed and how much mRNA (and obviously therefore protein) is produced
  2. Pax6
    this is a transcription factor, meaning the Pax6 gene codes for the Pax6 protein which itself is a Tx factor that controls the expression of OTHER genes that lead to the synthesis of OTHER, MORE DIFFERENT proteins! Wacky.
  3. transcription-control regions
    specific DNA sequences that serve as binding sites for Tx factors (aka repressor and activator proteins)
  4. there are no introns in:
    bacteria; bacteria are often organized into operons, which are genes that encode proteins all devoted to the same metabolic goal
  5. monocistronic mRNA
    An mRNA molecule that contains the genetic information to translate only ONE protein chain (polypeptide); this is the case for most eukaryotic mRNAs
  6. polycistronic mRNA
    mRNA that carries several open reading frames (ORFs), each of which is translated into a polypeptide; these proteins usually have a related function and their coding sequence is grouped/regulated together in an operon; bacteria

    -all genes in an operon are coordinately regulated: all activated or repressed to the SAME extent
  7. open reading frame (ORF)
    a DNA sequence that does not contain a stop codon in a given reading frame
  8. transcription (3 stages)
    initiation, elongation, termination (PLEASE MAKE THIS A TEST QUESTION)
  9. Image Upload 1
    PROMOTER is in the middle, RNA pol moves from left to right
  10. when Tx of a gene is repressed:
    corresponding mRNA end encoded prtotein(s) are synthesized at a low rate

    (opposite is true for when Tx of a gene is activated)

    -in bacteria, repression and activation of certain genes are mostly a response to environmental changes; so the organism can change what it uses its energy for accordingly
  11. lac operon (positive and negative control; inducible system)

  12. RNA pol (talking about II in this instance)
  13. -catalyzes RNA synthesis
    • -to attach to DNA strand of interest needs to associate with sigma factor
  14. holoenzyme
    complex of RNA pol II and the sigma factor it's bound to; together, the complex can find the promoter and transcribe the gene (generally just the complete complex containing all the subunits needed for activity)
  15. sigma factors
    • recognize the promoter SPECIFICALLY the consensus sequences at positions -35 and -10; once it is bound to transcript it recruits and attaches polymerase to itself
    • -is released after pol has transcribed a few 10's of bp's
    • -acts as an initiation factor required for initiation but NOT elongation

    • -these factors are for bacterial translation and here are some different kinds!
    • 70: attaches to most genes, promoter consensus:
    • 10 TATAAT
    • 35 TTGACA

    • 32: attaches to heat shock genes?
    • 28: motility
    • 38: be stationary
    • 54: NITROGEN metabolism
  16. translation termination
    Image Upload 2
    • -the main way to end transcription is to destabilize RNA pol's attachment to the DNA transcript
    • at the end of a coding sequence there are often times sequences in the DNA that when translated into RNA cause the RNA strand to associate with each other (aka a stem loop is formed because the sequences are complementary)
    • -after the stem loop there is often a repeat of U's which are unstable for the pairing of pol to DNA because of all the H-bonds (or A's, just in in general a repeat of U's T's or A's is more unstable than G's or C's because those are connected via 3x H-bonds)
  17. tryptophan synthesis is controled by negative regulation (also called a repressive system):
    • -tryptophan: amino acid used for the synthesis of many proteins
    • -trp operon: bacterial genome that codes for enzymes that function to make tryptophan; these enzymes participate in metabolic pathway in which end product = TRYPTOPHAN
    • -so if there is enough tryptophan for the cell at a certain point, there is no need for it to keep synthesizing more of it therefore trp operon transcription/translation STOPS (is repressed)
    • -repressor protein is ALWAYS expressed, meaning it's always present in the bacteria (whether it's bound to operon or not depends on other factors)
    • -go to a new card
  18. Trp Operon if there is NO tryptophan:
    • -respressor cannot bind to the operator site (aka it's inactive)
    • -pol II can bind to the operon and begin Tx of the gene
  19. Trp Operon if there is excess tryptophan:
    • -excess tryptophan binds to the respressor, converting it from its inactive to its ACTIVE form
    • -it can now bind to the operator site of the operon
    • -this prevents pol II from binding and subsequently Tx-ing the gene
    • -tryptophan acts as a co-repressor of the trp operon
  20. lac operon (inducible system that uses both positive and negative control)
    -encodes 3 enzymes necessary for the breakdown of lactose (sugar in milk) [so if there IS no lactose then the operon isn't expressed, because that'd be a waste of energy]

    • 1) lacZ - makes B-galactosidase (enzyme)
    • 2) lacY - makes permease (transporter)
    • 3) lacA - makes transacetylase (aids transporter)

    • -additional relevant genes: lacI makes the repressor and the operator sequence is where the repressor binds
    • -is always off unless inDUCED to be turned on
  21. Image Upload 3
    NO lactose present
    • -without lactose, the repressor (lacI) is BOUND to the operator, which overlaps the Tx start site
    • -no Tx of lac operon
    • -etc etc
  22. LACTOSE and gluose present
    • -if there is lactose (acts as an inducer) present, then it binds to the allosteric site of the repressor, changing its conformation and making it dissociate from the DNA
    • -pol II can then bind to the promoter and Tx occurs
    • -HOWEVER, if there's glucose present, the rate of Tx for the lac operon is very low (this is b/c the -10 and -35 sites where sigma70 binds differ from its ideal binding sites)
  23. only lactose present
    • -once glucose sources have been depleted, e.coli cells respond by synthesizing cAMP
    • -cAMP (small regulatory molecule) then binds to CAP protein, inducing a conformational change
    • -CAP/cAMP complex can now bind to CAP site in DNA
    • -the complex interacts with pol II bound at the prmoter and STIMULATES the rate of Tx
  24. lacI- and lacIs are mutants
    • -lacI- mutants cannot produce the repressor protein (that keeps the lac operon turned off), so there is constitutive expression of said operon
    • -lacI- CAN be canceled out via the insertion of a WT lacI+
    • - lacIs permanently represses the expression of the lac operon; meaning the repressor is ALWAYS bound to the operator even when inducer (lactose) binds to repressor; is dominant so can't be canceled out with WT copy of lacI+
  25. Image Upload 4
    how flexible is the DNA?
    • -y-axis can be read as though it shows the level of activation
    • -can use the graph to check the level of activation related to the distance between promoter and NtrC enhancer Optimal
    • -can see the distance associated with the highest level of activation (or concentration of protein) is 500 bp
    • -anything before and after is relatively less effective, especially after, b/c with increased distance between the two areas have a harder time finding each other
  26. NtrC protein (nitrogen regulatory protein C)
    stimlates Tx of of this glnA gene which encodes an enzyme that makes glutamine (amino acid, - (?) charged); interacts with sigma 54 (nitrogen break down) once it's associated with the promoter

    in fact, sigma 54 can't initiate the Tx UNTIL it associates with NtrC located far away
  27. DNA sense strand
    DNA strand is a sense strand if its sequence is the same as that of a mRNA copy that is translated into protein (only difference is that T's are U's)
  28. DNA antisense strand
    DNA on the strand opposite the sense strand (meaning corresponding mRNA is the opposite of every base in this strand)
Card Set
Meeting 13
7.0 7.1