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. 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. 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. 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 is an inducible operon that utilizes lactose as an energy source and is activated when glucose is low and lactose is present.
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. 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. 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. 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.
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.
Learning Objectives Explain the relationship between structure and function of an operon and the ways in which repressors regulate gene expression. 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. As previously described, in the absence of a functional trpR protein, the trp-sensitive negative feedback loop fails. TrpR mutants continue to produce trp in the presence of trp.
Strangely, however, trpR mutants grown in the absence of trp make even more trp than wild-type cells starved for trp, suggesting the existence of a secondary mechanism for sensing trp levels Oxender et al.
This trpR-independent mechanism for sensing trp levels is an example of attenuation. Continued molecular analysis revealed that a region within the trp operon mRNA was responsible for attenuation. This transcribed regulatory region, called the leader of the mRNA and located upstream of all the codons for the trp enzyme genes, interfered with expression of the trp operon by causing premature termination at an attenuation site located between the operator and the coding regions of the genes of the trp operon.
The mRNA leader can assume different shapes, or conformations, each one stabilized by base pairing Figure 1. One of these two conformations allows the rest of the operon to be transcribed and translated, but the other one does not. But how do these states depend upon tryptophan supply? The secret to this response lies in a tiny protein, or peptide , encoded by the leader. The leader peptide contains tryptophan codons, and when tryptophan is plentiful, it is translated easily.
This leads to the mRNA pairing that prevents transcription and translation of the rest of the operon. However, if tryptophan is in short supply, the peptide's translation stalls. This allows the second shape of the base-paired leader to form, which permits transcription and translation to continue. Note the base pairing that occurs in the different structures. The pairing is not perfect—there are certain nucleotides that do not pair. However, enough nucleotide interactions are present to stabilize these secondary structures.
The leader's structure plays a central role in mediating attenuation. That is, in the presence of trp, the newly synthesized trp operon mRNA adopts a conformation that interferes with continued transcription. Conversely, in the absence of trp, this conformation changes, allowing read-through. While repression of genes that are not needed provides clear survival benefits, a mechanism must exist for overcoming repression. Ideally, this mechanism should be responsive to cues to instigate situation-appropriate changes in gene expression.
In the case of the trp operon, the ligand tryptophan is required for the repressor to work repressible negative regulation.
But other operons respond to the presence of their small molecule signal ligand; that is, they are negatively regulated by a repressor protein, but they are inducible i. For example, repression of the lac operon by its repressor, called lacI, is inhibited by the ligand allolactose, to which the repressor protein directly binds.
Thus, lactose, from which allolactose is formed, induces the expression of the lac operon and of genes required for lactose metabolism. In the absence of lactose in the environment, the lac operon is transcribed at very low levels Figure 2. However, when lactose appears in the environment, a molecule produced from it allolactose can bind to the repressor lacI protein , thereby causing a conformational change. Note that there is a short period before the operon is fully expressed and the cell is fully able to metabolize available lactose.
This brief delay from basal expression to induced expression is called induction. Experiments by F. Jacob and J. Monod provided much of our foundational knowledge of the mechanisms of lactose metabolism in bacteria. In their research, Jacob and Monod noted that the lacI repressor, formed by a tetramer of the protein encoded by the lacI gene, binds to specific nucleotides in the operator lacO.
When that O sequence is mutated, the repressor can no longer bind, leaving the entire operon induced or "unrepressed. Thus, there is no induction time, as described in Figure 1.
When investigators tried to rescue this phenotype by adding a wild-type copy of the operon to the bacteria, they were unable to change the behavior of the endogenous mutated operon. Here, the researchers placed the wild-type O c operon on a plasmid that was separate from the bacterial chromosome , and both were present in the same cells.
Even when a wild-type copy was present in the cells and there was no lactose present, the cells expressed the lac operon, so the mutant O c was dominant. This suggested that the operator region controls only the genes adjacent to it, on the same piece of DNA.
In other words, the operator functions in a cis-dominant fashion. The case of the lacI repressor mutant, denoted lacI - , was quite different. Constitutive expression of the operon is also seen in lacI - cells.
But, contrary to O c mutants, the lacI - phenotype can be overcome by the addition of a wild-type lacI gene on a plasmid. This is because the wild-type lacI repressor protein is made correctly from the gene encoded by the plasmid.
The wild-type lacI protein can then bind to any lac operon operator sequence , including the endogenous version; thus, the repressor can act in trans. Because the wild-type lacI can rescue lacI - , the mutant version is recessive. In the case of a third mutant, lacI s , the result is a repressor that is constitutively bound to the operator.
Normally, the repressor protein has two conformations, or shapes. In one conformation, it is bound to the operator. When lactose is present, however, the lactose binds to the repressor, causing a change in conformation, and releasing the repressor from the operator. In lacI s mutants, the binding site for lactose is lost in the repressor protein.
As a result, no matter how much lactose is in the system, the operon stays in the "off" state. Moreover, if wild-type lacI is added on a plasmid, it cannot rescue this mutant. Thus, the mutation is dominant. Interestingly, the relatively simple mechanisms of gene expression in prokaryotic cells, as exemplified by the trp and lac operons, provide insight into several general principles involved in regulation in eukaryotes.
For example, specific sequences in DNA serve as binding sites for specific proteins that modulate the binding of RNA polymerase, the enzyme required for mRNA transcription. These operator sequences in DNA act in cis ; in other words, they control the expression of genes on the same contiguous piece of DNA, generally in fairly close proximity.
In contrast, the proteins that bind those sites act in trans; this means they can be produced by a gene elsewhere in the genome and act wherever the consensus sequence is located. Furthermore, the ability of E.
0コメント