Biochemistry I Fall Term, 2000

 October 16, 2000

Lecture 20: Transcription Regulation

Assigned reading in Campbell: Chapter 8.8-8.11

Key Terms:
lac Repressor & operator
CAP-cAMP activation
Closed & open promoter complexes
Initiation frequency
Similarities to Michaelis-Menten kinetics 		Exon & intron
Splicesome
TATA box (binding protein)
Ribozyme
 

Take the Review Quiz on Lecture 20 concepts.

These notes continue the outline from Lecture 19.

3. Control of Transcription in Prokaryotes.
  A. Expanded diagram of the lac promoter-operator region (PO).

Binding sites for the proteins are indicated with arrows. mRNA synthesis starts within the PO region and extends into the Z, Y, and A genes.
  B. Basic mechanism of RNA chain initiation.
R = holoenzyme.               P = promoter.
RPc = "closed complex".    RPo = "open complex".
KB = binding constant for RPc (M-1 ).
kf = rate constant for RPo formation from RPc (sec-1).
  C. RNA chain initiation frequency.
  1. Measurements in vivo: 10 RNA chains/min to <1 chain/hr.
  2. Measurements in vitro:
    a) Several different activity assays have been used to determine the time required to form the active open complexes in vitro. These data are then analyzed using the following equations:

    kobs is the rate constant for RPo formation. (These values come from the activity assays.)
    1/kobs = tobs , the average time required to form open complexes.
    Eqn. (1) shows that kobs approaches a maximum (kf) at saturating [R].
    Eqn. (2) is the reciprocal of eqn. (1); it predicts a linear plot of tobs vs. 1/[R].

Examples of both types of graph (for the same data):

The Y-intercept of the reciprocal plot is 1/kf; the intercept/slope = KB.
For the above data, KB = 107 M-1; kf = 1.0 min-1.

b) Both KB and kf differ for different promoters.
  1. KB : the range is from 106 M-1 to 109 M-1.
  2. kf : the range is from 10-4 sec-1 to 10-1 sec-1.
  3. tobs : varies from 10 sec to > 30 min.
    The times measured in vitro correlate well with in vivo frequencies.
c) KB and kf depend on the promoter sequence.
  1. bp changes away from consensus decrease KB or kf or both.
  2. bp changes toward consensus increase KB or kf or both.
  3. T.H.L.: "consensus is best"
d) Activators increase KB or kf or both.
  1. CAP-cAMP increases KB ~ 100-fold at lacP.
  2. lcI protein increases kf ~ 10-fold at the lPRM promoter.
  3. Other activators increase KB and kf.
e) Repressors decrease KB or kf or both.
  1. lacRep completely blocks productive RNA polymerase binding.
  2. Other repressors only decrease KB, i.e. as competitive inhibitors of RNA polymerase binding.

  D. Correspondence between measurements of vo for a Michaelis-Menten enzyme mechanism and kobs for RNA chain initiation. The table shows the experimental parameters, measured values, and the constants obtained from each type of biochemical analysis.

Michaelis- Menten RNA Chain Initiation
E P Use [P] << [R].
[S] [R] Determine the concentration dependence.
vo kobs Measure the rates of RPo formation.
Km 1/KB Find [R] at the half-maximal rate;
Vmax kf and find the maximal rate of RPo formation.
Vmax/Km KBkf "promoter strength" (M-1 sec-1)

Thus, the controlling step in mRNA synthesis (and therefore most of gene expression) can be understood using the same reasoning and tools we have used for simpler enzyme systems.


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