An operon is a functional unit of DNA found primarily in bacteria, consisting of a cluster of genes under the control of a single regulatory region. The trp operon in the bacterium Escherichia coli is a classic example, containing the instructions for making the enzymes needed to synthesize the amino acid tryptophan.
Tryptophan is a necessary building block for bacterial proteins, but synthesizing it requires significant cellular energy. Therefore, the trp operon is designed to be active only when external tryptophan levels are low, conserving resources when the amino acid is readily available. The operon’s five structural genes (trpE, trpD, trpC, trpB, trpA) are transcribed together into a single messenger RNA molecule. This mRNA is then translated to produce the full set of biosynthetic enzymes.
Dual Regulation: Repression and Attenuation
The trp operon employs two mechanisms to achieve control over tryptophan synthesis. The first mechanism is repression, which acts as the primary switch to prevent transcription from starting. This system involves a protein encoded by a separate gene called trpR, which produces an inactive repressor molecule.
When tryptophan is abundant, it binds to this repressor protein, acting as a co-repressor and changing the protein’s shape to an active form. The activated repressor then binds directly to the operon’s operator region, blocking RNA polymerase. This initial layer of control can reduce the expression of the trp operon by approximately 70-fold, effectively shutting down the pathway when tryptophan is plentiful.
A second, more subtle mechanism called attenuation provides a layer of fine-tuning that acts even after transcription has begun. Attenuation is a process of premature transcriptional termination. This mechanism is responsible for an additional 10-fold reduction in gene expression, bringing the total regulation to a 700-fold decrease when the amino acid is not needed. Attenuation is uniquely possible in bacteria because transcription and translation occur simultaneously in the cytoplasm.
The TrpL Leader Sequence: Attenuation’s Genetic Switch
The genetic element responsible for the attenuation mechanism is a short regulatory sequence known as the Tryptophan Leader, or trpL. The trpL sequence is located on the DNA after the operator and before the first structural gene, trpE. When the trp operon is transcribed, the trpL sequence is the first part of the messenger RNA molecule to be synthesized.
The resulting trpL mRNA transcript is about 162 nucleotides long and contains instructions for a leader peptide. Within this coding region, a pair of adjacent codons specific for tryptophan (Trp-Trp) is found. These two codons are the sensor elements of the attenuation system, as their translation speed depends directly on cellular tryptophan availability.
The trpL mRNA sequence is organized into four distinct segments, labeled Regions 1, 2, 3, and 4, which possess complementary base pairs. These regions can pair with each other to form alternative, mutually exclusive stem-loop structures. Critically, Region 3 can pair with either Region 2 or Region 4, and the choice between these two pairings determines whether transcription will continue or prematurely terminate.
How Tryptophan Levels Control Transcription Termination
The operation of the attenuation mechanism hinges on the speed of the ribosome as it translates the trpL leader peptide while the RNA polymerase is still transcribing the DNA. The availability of charged tRNA molecules carrying tryptophan dictates whether the ribosome moves quickly or stalls.
High Tryptophan Levels
When tryptophan concentration is high, the specific charged tRNA molecules are abundant. As the ribosome begins translating the trpL leader sequence, it encounters the two adjacent tryptophan codons in Region 1 and is able to insert the amino acids quickly without hesitation. This rapid translation causes the ribosome to move swiftly past Region 1 and cover Region 2 before the RNA polymerase has finished transcribing Region 4.
With Region 2 physically shielded by the fast-moving ribosome, Region 3 is forced to pair with Region 4, forming the 3-4 stem-loop structure. This 3-4 hairpin is known as the terminator loop. Once this terminator hairpin forms, the RNA polymerase dissociates, and transcription stops prematurely before the structural genes for tryptophan synthesis are copied.
Low Tryptophan Levels
When the cellular concentration of tryptophan is low, the ribosome reaches the tandem tryptophan codons in Region 1 and must pause and wait for a charged tRNA-tryptophan to become available. This stalling of the ribosome at Region 1 leaves Region 2 exposed to pair with Region 3.
The pairing of Region 2 with Region 3 creates the 2-3 stem-loop, which is called the anti-terminator hairpin. The formation of this anti-terminator structure prevents the formation of the 3-4 terminator loop. Because the termination signal is absent, the RNA polymerase continues transcribing past the trpL sequence and copies the five structural genes, ultimately leading to the production of the enzymes needed to manufacture the scarce tryptophan.