How the trp repressor controls expression gene expression. Feedback inhibition & attenuation.

Key points:

  • The trp operon, found in E. coli bacteria, is a group of genes that encode biosynthetic enzymes for the amino acid tryptophan.
  • The trp operon is expressed (turned "on") when tryptophan levels are low and repressed (turned "off") when they are hight.
  • The trp operon is regulated by the trp repressor. When bound to tryptophan, the trp repressor blocks expression of the operon.
  • Tryptophan biosynthesis is also regulated by attenuation (a mechanism based on coupling of transcription and translation).

What is the trp operon?

Bacteria such as Escherichia coli (a friendly inhabitant of our gut) need amino acids to survive—because, like us, they need to build proteins. One of the amino acids they need is tryptophan.
If tryptophan is available in the environment, E. coli will take it up and use it to build proteins. However, E. coli can also make their own tryptophan using enzymes that are encoded by five genes. These five genes are located next to each other in what is called the trp operon.
The short answer is that an operon is a set of genes that are transcribed under control of a single promoter, resulting in one long mRNA that contains coding sequences for multiple genes. The operon includes not only the genes, but also the regulatory DNA sequences that control their expression (including the promoter and binding sites for any repressor or activator proteins).
To review the basic properties of operons step-by-step, see the overview of gene regulation in bacteria article.
If tryptophan is present in the environment, then E. coli bacteria don't need to synthesize it, so transcription of the genes in the trp operon is switched "off." When tryptophan availability is low, on the other hand, the operon is switched "on," the genes are transcribed, biosynthetic enzymes are made, and more tryptophan is produced.

Structure of the trp operon

The trp operon includes five genes that encode enzymes needed for tryptophan biosynthesis, along with a promoter (RNA polymerase binding site) and an operator (binding site for a repressor protein). The genes of the trp operon are transcribed as a single mRNA.
Diagram of the trp operon. First, we see an E. coli bacterium with a circular chromosome. We zoom in on a small portion of the chromosome and see that the DNA is that of the trp operon.
From left to right, the operon contains a promoter (where RNA polymerase binds), and within the right end of the promoter, an operator (where a repressor binds). There are some additional regulatory sequences, not labeled in this diagram, and then five coding sequences: trpE, trp_D, _trpC, trpB, and trpA.
The operon is transcribed to produce a single mRNA that contains the coding sequences of all five of the genes.
The coding sequences in the mRNA are translated separately, each one producing a protein. These proteins are enzymes (or enzyme subunits) needed for tryptophan biosynthesis.

Turning the operon "on" and "off"

What does the operator do? This stretch of DNA is recognized by a regulatory protein known as the trp repressor. When the repressor binds to the DNA of the operator, it keeps the operon from being transcribed by physically getting in the way of RNA polymerase, the transcription enzyme.
The trp repressor protein is encoded by a gene called trpR. This gene is not part of the trp operon, and it's located elsewhere on the bacterial chromosome, where it has its own promoter and other regulatory sequences.
The trp repressor does not always bind to DNA. Instead, it binds and blocks transcription only when tryptophan is present. When tryptophan is around, it attaches to the repressor molecules and changes their shape so they become active. A small molecule like trytophan, which switches a repressor into its active state, is called a corepressor.
High tryptophan: The tryptophan binds to the trp repressor and causes it to change shape, converting into its active (DNA-binding) form. The trp repressor with the bound tryptophan attaches to the operator, blocking RNA polymerase from binding to the promoter and preventing transcription of the operon.
When there is little tryptophan in the cell, on the other hand, the trp repressor is inactive (because no tryptophan is available to bind to and activate it). It does not attach to the DNA or block transcription, and this allows the trp operon to be transcribed by RNA polymerase.
Low tryptophan: trp repressor is not bound to tryptophan (since there is no tryptophan) and is thus in its inactive state (does not bind to the DNA of the operator). This allows RNA polymerase to bind to the operator and transcribe the operon.
In this system, the trp repressor acts as both a sensor and a switch. It senses whether tryptophan is already present at high levels, and if so, it switches the operon to the "off" position, preventing unnecessary biosynthetic enzymes from being made.

More trp operon regulation: Attenuation

Depending on the class you're taking, or on your own interests, you may also have heard about another form of trp operon regulation called attenuation.
Like regulation by the trp repressor, attenuation is a mechanism for reducing expression of the trp operon when levels of tryptophan are high. However, rather than blocking initiation of transcription, attenuation prevents completion of transcription.
When levels of tryptophan are high, attenuation causes RNA polymerase to stop prematurely when it's transcribing the trp operon. Only a short, stubby mRNA is made, one that does not encode any tryptophan biosynthesis enzymes. Attenuation works through a mechanism that depends on coupling (the translation of an mRNA that is still in the process of being transcribed).
To understand attenuation, let's zoom in on a region of the trp operon that we skimmed over in the sections above. This section lies between the operator and the first gene of the operon and is called the leader. The leader encodes a short polypeptide and also contains an attenuator sequence. The attenuator does not encode a polypeptide, but when transcribed into mRNA, it has self-complementary sections and can form various hairpin structures.
Image showing the location of the leader. The leader comes after the promoter and operator, but before the trpE gene. From left to right, the leader DNA contains four marked segments labeled 1-4. Segment 1 encodes the leader polypeptide. Segments 2-4 are part of the attenuator.
Once RNA polymerase has started transcribing the operon, a ribosome can attach to the still-forming transcript and begin translating the leader region. The polypeptide encoded by the leader is short, just 14 amino acids long, and it includes two tryptophan (Trp) residuesstart superscript, 1, end superscript. The tryptophans are important because:
  • If there is plenty of tryptophan, the ribosome won't have to wait long for a tryptophan-carrying tRNA, and will rapidly finish the leader polypeptide.
  • If there is little tryptophan, the ribosome will stall at the Trp codons (waiting for a Trp-carrying tRNA) and will be slow to finish translation of the leader.
Why does it matter if the ribosome translates the leader quickly or slowly? As mentioned above, the leader is followed by an attenuator region, which (in its mRNA form) can stick to itself to form different hairpin structures. One structure includes a transcription termination signal, while the other does not end termination (and in fact, prevents formation of the terminator hairpin)start superscript, 2, end superscript.
  • If the ribosome translates slowly, it will pause, and its pausing causes formation of the antiterminator (non-terminating hairpin). This hairpin prevents formation of the terminator and allows transcription to continue.
    Low Trp levels: When there is not much tryptophan available in the cell, the ribosome will stall at the Trp codons while translating the short polypeptide at the start of the leader. This stalling causes regions 2 and 3 to associate with one another in a hairpin. This hairpin, called an antiterminator hairpin, prevents the terminator hairpin (regions 3 and 4 paired up) from forming. Termination does not occur and RNA polymerase continues transcribing, producing a transcript that includes the trpE-trpA genes.
  • If the ribosome translates quickly, it will fall off the mRNA after translating the leader peptide. This allows the terminator hairpin and an associated hairpin to form, making RNA polymerase detach and ending transcription.
    High Trp levels: The ribosome does not stall at the Trp codons while synthesizing the leader polypeptide, because Trp is abundant (and there are thus plenty of Trp-carrying tRNAs around). Instead, the ribosome quickly synthesizes the leader polypeptide, reaches the stop codon, and detaches from the mRNA. This leaves regions 1 and 2 free to pair up, at which point regions 3 and 4 will also pair up and form a terminator hairpin. The terminator hairpin causes RNA polymerase to detach from the DNA and from the transcript, ending termination. A short mRNA consisting of the leader region is all that gets produced; the trpE-trpA genes are never transcribed.
This mechanism may be complex, but the result is pretty straightforward. When tryptophan is abundant, the ribosome moves quickly along the leader, the terminator hairpin forms, and transcription of the trp operon ends. When tryptophan is scarce, the ribosome moves slowly along the leader, the non-terminator hairpin forms, and transcription of the trp operon continues.
In other words, the logic is of attenuation is the same as that of regulation by the trp repressor. In both cases, high levels of tryptophan in the cell shut down the expression of the operon. This makes sense, since high levels of tryptophan mean that the cell does not need to make more biosynthetic enzymes to produce additional tryptophan.

Attribution:

This article is a modified derivative of "Prokaryotic gene regulation," by OpenStax College, Biology, CC BY 4.0. Download the original article for free at http://cnx.org/contents/76b4a074-d223-4ad9-8d9e-4114c74f492c@5.
The modified article is licensed under a CC BY-NC-SA 4.0 license.

Works cited:

  1. Yanofsky, C. (2007). RNA-based regulation of genes of tryptophan synthesis and degradation, in bacteria. RNA, 13(8), 1143. http://dx.doi.org/10.1261/rna.620507.
  2. Yanofsky, C. (2007). RNA-based regulation of genes of tryptophan synthesis and degradation, in bacteria. RNA, 13(8), 1144. http://dx.doi.org/10.1261/rna.620507.

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