Prokaryotic Gene Regulation

The trp Operon: A Repressor Operon

The trp operon is a repressor operon that is either activated or repressed based on the levels of tryptophan in the environment.

Learning Objectives

Explain the relationship between structure and function of an operon and the ways in which repressors regulate gene expression

Key Takeaways

Key Points

  • The operator sequence is encoded between the promoter region and the first trp-coding gene.
  • The trp operon is repressed when tryptophan levels are high by binding the repressor protein to the operator sequence via a corepressor which blocks RNA polymerase from transcribing the trp-related genes.
  • The trp operon is activated when tryptophan levels are low by dissociation of the repressor protein to the operator sequence which allows RNA polymerase to transcribe the trp genes in the operon.

Key Terms

  • repressor: any protein that binds to DNA and thus regulates the expression of genes by decreasing the rate of transcription
  • operon: a unit of genetic material that functions in a coordinated manner by means of an operator, a promoter, and structural genes that are transcribed together

The trp Operon: A Repressor Operon

Bacteria such as E. coli need amino acids to survive. Tryptophan is one such amino acid that E. coli can ingest from the environment. E. coli can also synthesize tryptophan using enzymes that are encoded by five genes. These five genes are next to each other in what is called the tryptophan (trp) operon. If tryptophan is present in the environment, then E. coli does not need to synthesize it; the switch controlling the activation of the genes in the trp operon is turned off. However, when tryptophan availability is low, the switch controlling the operon is turned on, transcription is initiated, the genes are expressed, and tryptophan is synthesized.

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The trp operon: The five genes that are needed to synthesize tryptophan in E. coli are located next to each other in the trp operon. When tryptophan is plentiful, two tryptophan molecules bind the repressor protein at the operator sequence. This physically blocks the RNA polymerase from transcribing the tryptophan genes. When tryptophan is absent, the repressor protein does not bind to the operator and the genes are transcribed.

A DNA sequence that codes for proteins is referred to as the coding region. The five coding regions for the tryptophan biosynthesis enzymes are arranged sequentially on the chromosome in the operon. Just before the coding region is the transcriptional start site. This is the region of DNA to which RNA polymerase binds to initiate transcription. The promoter sequence is upstream of the transcriptional start site. Each operon has a sequence within or near the promoter to which proteins (activators or repressors) can bind and regulate transcription.

A DNA sequence called the operator sequence is encoded between the promoter region and the first trp-coding gene. This operator contains the DNA code to which the repressor protein can bind. When tryptophan is present in the cell, two tryptophan molecules bind to the trp repressor, which changes shape to bind to the trp operator. Binding of the tryptophan–repressor complex at the operator physically prevents the RNA polymerase from binding and transcribing the downstream genes.

When tryptophan is not present in the cell, the repressor by itself does not bind to the operator; therefore, the operon is active and tryptophan is synthesized. Because the repressor protein actively binds to the operator to keep the genes turned off, the trp operon is negatively regulated and the proteins that bind to the operator to silence trp expression are negative regulators.

Catabolite Activator Protein (CAP): An Activator Regulator

When glucose levels decline in E. coli, catabolite activator protein (CAP) is bound by cAMP to promote transcription of the lac operon.

Learning Objectives

Explain how an activator works to increase transcription of a gene

Key Takeaways

Key Points

  • Catabolite activator protein (CAP) must bind to cAMP to activate transcription of the lac operon by RNA polymerase.
  • CAP is a transcriptional activator with a ligand-binding domain at the N-terminus and a DNA -binding domain at the C-terminus.
  • cAMP molecules bind to CAP and function as allosteric effectors by increasing CAP’s affinity to DNA.

Key Terms

  • RNA polymerase: a DNA-dependent RNA polymerase, an enzyme, that produces RNA
  • operon: a unit of genetic material that functions in a coordinated manner by means of an operator, a promoter, and structural genes that are transcribed together
  • promoter: the section of DNA that controls the initiation of RNA transcription

Catabolite Activator Protein (CAP): An Activator Regulator

Just as the trp operon is negatively regulated by tryptophan molecules, there are proteins that bind to the operator sequences that act as a positive regulator to turn genes on and activate them. For example, when glucose is scarce, E. coli bacteria can turn to other sugar sources for fuel. To do this, new genes to process these alternate genes must be transcribed. This type of process can be seen in the lac operon which is turned on in the presence of lactose and absence of glucose.

When glucose levels drop, cyclic AMP (cAMP) begins to accumulate in the cell. The cAMP molecule is a signaling molecule that is involved in glucose and energy metabolism in E. coli. When glucose levels decline in the cell, accumulating cAMP binds to the positive regulator catabolite activator protein (CAP), a protein that binds to the promoters of operons that control the processing of alternative sugars, such as the lac operon. The CAP assists in production in the absence of glucose. CAP is a transcriptional activator that exists as a homodimer in solution, with each subunit comprising a ligand-binding domain at the N-terminus, which is also responsible for the dimerization of the protein and a DNA-binding domain at the C-terminus. Two cAMP molecules bind dimeric CAP with negative cooperativity and function as allosteric effectors by increasing the protein’s affinity for DNA. CAP has a characteristic helix-turn-helix structure that allows it to bind to successive major grooves on DNA. This opens up the DNA molecule, allowing RNA polymerase to bind and transcribe the genes involved in lactose catabolism. When cAMP binds to CAP, the complex binds to the promoter region of the genes that are needed to use the alternate sugar sources. In these operons, a CAP-binding site is located upstream of the RNA-polymerase-binding site in the promoter. This increases the binding ability of RNA polymerase to the promoter region and the transcription of the genes. As cAMP-CAP is required for transcription of the lac operon, this requirement reflects the greater simplicity with which glucose may be metabolized in comparison to lactose.

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Catabolite Activator Protein (CAP) Regulation: When glucose levels fall, E. coli may use other sugars for fuel, but must transcribe new genes to do so. As glucose supplies become limited, cAMP levels increase. This cAMP binds to the CAP protein, a positive regulator that binds to an operator region upstream of the genes required to use other sugar sources.

The lac Operon: An Inducer Operon

The lac operon is an inducible operon that utilizes lactose as an energy source and is activated when glucose is low and lactose is present.

Learning Objectives

Describe the components of the lac operon and their role in its function

Key Takeaways

Key Points

  • The lac operon contains an operator, promoter, and structural genes that are transcribed together and are under the control of the catabolite activator protein (CAP) or repressor.
  • The lac operon is not activated and transcription remains off when the level of glucose is low or non-existent, but lactose is absent.
  • The lac operon encodes for the genes needed to utilize lactose as an energy source.

Key Terms

  • operator: a segment of DNA to which a transcription factor protein binds
  • repressor: any protein that binds to DNA and thus regulates the expression of genes by decreasing the rate of transcription

The lac Operon: An Inducer Operon

A major type of gene regulation that occurs in prokaryotic cells utilizes and occurs through inducible operons. Inducible operons have proteins that can bind to either activate or repress transcription depending on the local environment and the needs of the cell. The lac operon is a typical inducible operon. As mentioned previously, E. coli is able to use other sugars as energy sources when glucose concentrations are low. To do so, the cAMP–CAP protein complex serves as a positive regulator to induce transcription. One such sugar source is lactose. The lac operon encodes the genes necessary to acquire and process the lactose from the local environment, which includes the structural genes lacZ, lacY, and lacA. lacZ encodes β-galactosidase (LacZ), an intracellular enzyme that cleaves the disaccharide lactose into glucose and galactose. lacY encodes β-galactoside permease (LacY), a membrane-bound transport protein that pumps lactose into the cell. lacA encodes β-galactoside transacetylase (LacA), an enzyme that transfers an acetyl group from acetyl-CoA to β-galactosides. Only lacZ and lacY appear to be necessary for lactose catabolism.

CAP binds to the operator sequence upstream of the promoter that initiates transcription of the lac operon. The lac operon uses a two-part control mechanism to ensure that the cell expends energy producing β-galactosidase, β-galactoside permease, and thiogalactoside transacetylase (also known as galactoside O-acetyltransferase) only when necessary. However, for the lac operon to be activated, two conditions must be met. First, the level of glucose must be very low or non-existent. Second, lactose must be present. If glucose is absent, then CAP can bind to the operator sequence to activate transcription. If lactose is absent, then the repressor binds to the operator to prevent transcription. If either of these requirements is met, then transcription remains off. The cell can use lactose as an energy source by producing the enzyme b-galactosidase to digest that lactose into glucose and galactose. Only when both conditions are satisfied is the lac operon transcribed, such as when glucose is absent and lactose is present. This process is beneficial and makes most sense for the cell as it would be energetically wasteful to create the proteins to process lactose if glucose were plentiful or if lactose were not available.

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The lac Operon: Transcription of the lac operon is carefully regulated so that its expression only occurs when glucose is limited and lactose is present to serve as an alternative fuel source.