Why have gene expression regulation?

• It simply would fail to have all genes always expressed.
• Different points of gene regulation in prokaryotes can be seen in Figure 21.1.
• Major types of regulation:
  • Repressible System: The end product of the system increases, which shuts off transcription of the coding region necessary for such synthesis. For example, if an amino acid concentration increases to a certain level, transcription necessary for that amino acid shuts off.
  • Inducible System: When a substance is present, genes are expressed, when the substance is gone, genes are no longer expressed.
  • See Figure 21.2.

Some prokaryotic genes code for one gene product. More interestingly, some genes share the same promoter and are expressed by the generation of a single transcript with several different starting places for the synthesis of more than one different protein. These genes are said to belong to a single operon.

The basic operon is conceptualized in Figure 21.3.

LAC Operon: The paradigm of inducible prokaryotic gene regulation.

• Lactose can be broken down into glucose and galactose.
• This is done by the enzyme B-galactosidase (coded for by lacZ gene).
• There are very few molecules of this enzyme in normal cells when no lactose is present. When lactose is added to the medium, the concentration of this enzyme increases rapidly.
• Also, two other enzymes increase: B-galactoside permease (coded for by the lacY gene), B-galactoside acetyltransferase (coded for by lacA).
• The permease is involved in transport of lactose across the membrane.
• The transferase protects other intracellular galactosides from the activity of B-galactosidase. See Figure 21.4.
• The three genes are all induced together, but also are located next to each other on the chromosomes. In fact, they are coded for on the same polycistronic mRNA.
• A regulatory molecule called a repressor is coded for by the lacI gene. It too is located adjacent to the other three but its regulation is totally independent to the other lac genes. The repressor interferes with the transcription of genes involved in lactose metabolism.

The OPERATOR is a site on the DNA that is recognized by the repressor protein. When this site is bound by the repressor, RNA polymerase rarely binds the promoter. This prevents transcription.

1. For expression of the genes of the lac operon, the repressor must be absent.

2. The repressor is an allosteric protein. This means it changes its shape, when it bind certain molecules. The lac operon repressor is a protein with 4 subunits. Two of the subunits bind the operator, the other two subunits can bind the allolactose. Allolactose is a lactose analog, which for the lac operon, is the inducer.

3. When the allolactose binds the repressor, the repressor's affinity for the operator sequence decreases drastically.

4. The genes of the lac operon are then expressed.

The allolactose is created by B-galactosidase; it is an alternative product to the normal cleaved one. The B-galactosidase that creates this inducer is at very low levels in the cell when the operon is being repressed. See Figure 21.5.

The term constitutive means always expressed. A constitutive mutant is one where the genes are always expressed; when in the wild-type, the expression of the same is under regulation.

The lac repressor protein is coded for by a gene that is constitutively expressed.

Some mutants of the lac operon make it so the operon is constitutively expressed. Having a defective or a lack of the repressor protein would cause this. Some mutations of the operator sequence does so also.

The lac operon was studied using mutants; some were partial diploids. See Figure 21.6.

Glucose is the preferred energy substrate to lactose in E.coli. The cell will use glucose in preference to lactose, when both are present. The mechanism used is called catabolite repression.

1. It involves a catabolite activator protein (CAP), to control transcription of certain operons.

2. When there is no glucose available, the CAP binds cAMP and then binds to a region of the promoter of operons that have CAP sites.

3. When this region of the promoter is bound, RNA polymerase has a greater ability to bind and produce transcripts. See Figures 21.8 and 21.9.

1. The repressor in this case is the product of the trpR gene.

2. The end product of the operon, tryptophan, is the corepressor.

3. When tryptophan is bound by the repressor, the repressor binds the operator, which shuts off transcription. When no tryptophan is bound to the repressor, the repressor disassociates from the operator, transcription then begins. See Figure 21.10.

1. The trp operon also has an attenuator region.

2. It is located between the operator and the first structural gene of the operon.

3. The mRNA transcript from this region is called a leader transcript.

4. This mRNA transcript can form three different stem-loop structures, from 4 different regions.

5. 1-2 and 3-4 can form 2 stem-loop structures; 2-3 can form another stem-loop, exclusive to the other 2. It is an either, or.

6. A peptide is coded for by the leader mRNA sequence; two of the amino acids are tryptophan.

7. If excess tryptophan, then there are plenty of trp - tRNAs. If little or no tryptophan, then very few trp - tRNAs.

8. If promoter region is active (no bound repressor/corepressor), and there are trp - tRNAs, then translation occurs on the leader mRNA sequence. Translation proceeds through the codon that codes for tryptophan.

9. If it reads through the trp codon, the stem-loop 3-4 forms, which has the configuration of a terminator, called an attenuator stem.

10. If little trp-tRNA is available, the ribosomes stall at the trp codon, create a 2-3 stem, which does not terminate the transcription. The stall allows the RNA polymerase to continue down the DNA strand far enough away so that it is no longer affected by the stem loop terminator when it does eventually form. See Figure 21.11, 21.12 and 21.13.

• To be covered in class

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