Regulation of Genes
Classday 10

1. Global Control ("magic spot") (Mike Cassell and Mike Holmes) By "global" is meant that the whole chromosome is turned "on" or "off". This is what is called the "stringent response" and is a life-saver for many organisms in times of privation. When food supplies or some other factor is in short supply, the cells merely turn "off" rather than struggle onwards and risk depleting all of their energy stores and die. Better to shut down to dormancy to await a better day.
   1. Translation forming polypeptide. The following is the normal pathway. Notice what energy drives the reaction (hint: it ends with "-TP"!), and what the substrates and products are.
      

   2. Strangled bacteria make MS. Anything that prevents the formation of activated tRNA's (any one of them!) results in the triggering of stringency. Examples are the depletion of an amino acid from the medium. Even if the cell must then switch on existing genes to start the synthesis of that amino acid, the cell is triggered to turn off and run in low gear while that gene is ramped up into production. Another trigger for the making of MS (magic spot = G4P) is the sudden depletion of dissolved oxygen. The cell, which is acclimated for growing in oxygen, suddenly finds none and ATP synthesis suddenly halts, and that halts the synthesis of activated amino acids and hence no synthesis of activated tRNA's. This effect is so rapid that the whole phenomenon was discovered by the mere pipetting a culture from one flask and putting it into another. Just those moments in the pipet allowed the aerobic bacteria to deplete the oxygen!

   3. The role of the ribosome in stringency (relA) The best monitor or quality control manager for whether or not any tRNA's are not activated is a component of the ribosome, which can be thought of as the work-bench upon which polypeptides are assembled. One of the "r-proteins" that constitute the smaller part of the prokaryotic ribosome is called the stringent factor, and it is the product of the gene called relA. It was so named because when there is a mutant in this gene, the cell is no longer stringent in its nutrition, but is "relaxed".

   4. The synthesis of MS (spoT) Here you see that SF "saw" the presence of a naked tRNA, and that meant the cell was in nutrient deprivation. So SF lept into action and converted a huge amount of GTP into G5P (guanosine pentaphosphate), which dephosphorylated by the product of a gene whimsically called spoT to G4P ("MS" = Magic Spot), which is an allosteric inhibitor of transcriptase. Magic Spot was so named because once the abovementioned pipetting was done, a huge amount of P-32 phosphate suddenly appeared as if by magic on a two-dimensional chromatogram - a place where no spot had ever before been seen. Indeed, within seconds, 99% of the available phosphate in the cell was found in this spot! Later it was found that MS masked another spot, one called phantom spot, which turned out to be G5P. Anyway, if transcriptase is inactivated, the cell screeches to an almost complete stop metabolically.
      

   5. Inactivation of DNA-dependent RNA polymerase (what's its trivial name?)

   6. Recovery This aspect has not been nearly so well studied, and the prevailing notions are that MS is a bit unstable and slowly hydrolyzes spontaneously, thus reactivating transcriptase. (ATP, by the way, has a halflife of about 15 minutes in water at room temperature.)

2. Course control
   1. Placement of the genes on the Chromosome
      Three major bits of evidence stand behind this idea:
      1. Later we will see how the line-up of genes on viral chromosomes affect their expression.

      2. This author showed that the genes governing the rate limiting enzymes of the synthesis of the many different amino acids were located at distances from E. coli's oriC commensurate with the concentration of the individual amino acids.
         

         You will notice in the bottom figure, above, that there are FOUR oriC's to each "terminus", and that the closer one moves toward the terminus, the number of copies of a gene approaches one. Thus the "gene dosage" smoothly goes from 4 ("early") to 1 ("late").

      3. This author then showed actual supporting evidence such that when the lac-operon was translocated to other positions on the E. coli chromosome, the amount of ß-galactosidase produced was also commensurate with the gene's closeness to oriC.

   2. Regulon

   3. Operon (know the lac-operon generally and more specifically!)

3. Fine control
   1. operators and promoters
      1. mutations (e.g.: UV5)

      2. CAP and catabolite repression
         Even more Nobel stuff! It was long known - the Pasteur Effect - that when cells were fed an overabundance of an energy source that they would slow down many of their processes. This was extremely puzzling. Finally it was determined that when there was an abundance of energy to make lots of ATP it was the excess ATP that inhibited further reactions. ATP is a catabolite, hence 'catabolite repression.' It works like this: many genes need a catabolite activating protein, CAP, to attach to the promoter region of their operons. CAP acts something like a doorman that hails a taxi for you. The taxi more easily sees where to stop because the doorman is in a distinctive uniform. When CAP affixes to the right DNA sequence, transcriptase more easily finds where to "sit down" and begin reading. But CAP requires a coenzyme to do its work properly - just like a doorman requires a special whistle! The coenzyme is cyclic-AMP. If there is cAMP around to help CAP, the gene is expressed. However if there is no cAMP around, the gene goes unnoticed. How is cAMP made?
         ATP --(adenyl-cyclase)--> cAMP
         However the substrate ATP is itself an allosteric inhibitor of this enzyme! Too much ATP, and no cAMP is made. This was discovered to be good: if glucose AND lactose are fed to an E.coli culture, the presence of glucose shuts down the lac-operon because the glycolysis of glucose makes a lot of ATP and hence no cAMP is made with CAP needs, which is in turn needed by the lac-operon for expression. Why turn on the lac-operon when there is enough ATP coming from consuming simpler glucose?!

         You might consider that the enzyme adenyl-cyclase has two ATP-binding sites: one is the enzymatic site for converting ATP to cAMP, and the other with a lower affinity binds ATP and distorts the shape of the enzyme so that the enzymatic site cannot work.

      3. Attenuators and Effectors are to be covered later.

   2. Allosteric effects (Jacob & Monod; Nobel Prize work) In the following cartoon, Pacman represents any one of thousands of different enzymes in the cell. In this case, suppose that it represents the lactose repressor protein, which binds to the DNA in the lac-operon at a very specific site known as the "operator" (lac-O). When it binds it blocks the site where transcriptase wants to begin reading the DNA, and thus effectively prevents the reading of that gene. That's why it is called a "repressor." However, sometimes the cell should read that gene and the repressor protein needs to be inactivated. Conceptually, this might be accomplished in two ways by the substrate (the sugar lactose, which needs to be degraded by the products of that gene). Pacman's mouth wants to clamp onto the DNA. But if his mouth is already filled with lactose, he cannot hang onto the DNA. This is called competitive inhibition because lactose can compete with the DNA for the "mouth." (A very small amount of lactose might not be enough to fill the mouth and prevent Pacman from latching onto the DNA.) Another way - and this is THE way that it works in the case of lac-repressor - is by having the lactose bind to the repressor protein somewhere else (a second site) that somehow prevents Pacman from latching onto the DNA. This is shown as lactose's being a patch over Pacman's eyes so that he cannot find the DNA. Another type of this "second site" or "allosteric" inhibition might be thought of as binding to Pacman's sides and tickling him so that in his laughter he cannot keep his mouth closed. In other words, an allosteric inhibitor distorts the shape of the protein so that it cannot do its job.
      

      Now if you really want to get confused with theory of enzyme inhibition read a chapter in a major biochemistry textbook in which these three types of inhibition are discussed: competitive, non-competitive and un-competitive inhibitions!