Tryptophan trp Operon

The tryptophan operon is responsible for the production of the amino acid tryptophan, whose synthesis occurs in 

Three states of the lac operon showing that lac mRNA is made only if cAMP-CRP is present and repressor is absent. 

Regulation of the trp operon is determined by the concentration of tryptophan when adequate tryptophan is present in the growth medium, there is no need for tryptophan biosynthesis. Transcription is turned off when a high concentration of tryptophan is present and is turned on when tryptophan is absent. The regulatory signal is the concentration of tryptophan itself and, in contrast with lactose, tryptophan is active in repression rather than induction. 

The trp operon has two levels of regulation—an on-off mechanism and a modulation system. The protein product of the trpR gene—the trp aporepressor—cannot bind to the operator in contrast with the lac repressor. However, if tryptophan is present, the aporepressor and the tryptophan molecule join together to form an active repressor complex 

When the trp operon is derepressed, which is usually the case unless the concentration of tryptophan in the medium is very high, the optimal concentration of tryptophan is maintained by a modulating system in which the enzyme concentration varies with the concentration of tryptophan. This modulation is effected by 

---

Organization of The tryptophan (trp) operon (Fig. 1) contains five structural genes encoding... 

Figure 13.2 E. coli tryptophan (trp) operon. o, operator L, leader a, attenuator E, D, C, B, A, structural genes. Numbers are base pairs.

The physiological signal controlling the lac and ara operons is the utilization of carbon sources for metabolic energy. In contrast, the tryptophan trp) operon is sensitive to the need for biosynthetic processes and is transcribed under conditions where intracellular concentrations of the amino acid tryptophan are below an optimal level for efficient protein synthesis. The trp operon consists of a promoter and operator region which controls the expression of a polycistronic mRNA encoding five proteins needed for tryptophan biosynthesis. 

The E. coli tryptophan (trp) operon (Fig. 28-19) includes five genes for the enzymes required to convert chorismate to tryptophan. Note that two of the enzymes catalyze more than one step in the pathway. The mRNA from the trp operon has a half-life of only about 3 min, allowing the cell to respond rapidly to changing needs for this amino acid. The Trp repressor is a homodimer, each subunit containing 107 amino acid residues (Fig. 

Provide an overview of the regulation of the tryptophan (trp) operon by attenuation. 

The Lac operon is but one example of the genetic adaptations which allow bacteria to respond to their environment. Other examples are to be found in amino acid metabolism, for example the TRP operon which regulates tryptophan metabolism. 

The enzymes of Trp-biosynthesis are only required if too little tryptophan is available to the bacteria from the growth medium. In such a case the Trp requirement is fulfilled by the cell s own Trp biosynthesis. If however, there is enough Trp supplied by the medium, then it is prudent to shut down the Trp operon. The sensor is the Trp concentration. The Trp repressor registers the current Trp concentration with the help of its own Trp binding site. If a great deal of Trp is present, then the Trp binding site of the repressor is occupied by Trp. The Trp repressor binds Trp with high affinity (Kd=10 -10 M), upon which transcription of the operon is then blocked. 

FIGURE 28-19 The trp operon. This operon is regulated by two mechanisms when tryptophan levels are high, (1) the repressor (upper left) binds to its operator and (2) transcription of trp mRNA is attenuated (see Fig. 28-21). The biosynthesis of tryptophan by the enzymes encoded in the trp operon is diagrammed at the bottom... 

Attenuation. A major mechanism of feedback repression, known as attenuation, depends not upon a repressor protein but upon control of premature termination. It was first worked out in detail by Yanofsky et al. for the trp operon of E. coli and related bacteria.184 186 Accumulation of tryptophan in the cell represses the trp biosynthetic operon by the action of accumulating tryptophanyl-tRNATlP, which specifically induces termination in the trp operon. Other specific "charged" arnino-acyl-tRNA molecules induce termination at other amino acid synthesis operons. 

Schematic diagram of the repressor control of trp operon expression. The trp promoter (P) and trp operator (O) regions overlap. The trp aporepressor is encoded by a distantly located trpR gene. L-Tryptophan binding converts the aporepressor to the repressor that binds at the operator locus. This complex prevents the formation of the polymerase-promoter complex and transcription of the operon that begins in the leader region (trpL). Only a fraction of the transcripts extends beyond the attenuator locus in the leader region. The regulation of this fraction is discussed in the text.

Model for attenuation in the trp operon, showing ribosome and leader RNA. (a) Where no translation occurs, as when the leader AUG codon is replaced by an AUA codon, stem-and-loop 3.4 is intact, and termination in the leader is favored. (b) Cells are selectively starved for tryptophan so that the ribosome stops prematurely at the tandem trp codons. Under these conditions, stem-and-loop 2.3 can form, and this is believed to lead to the disruption of stem-and-loop 3.4. (c) All amino acids, including excess tryptophan, are present so that stem-and-loop 3.4 is present. 

The trp operon contains a cluster of five structural genes associated with tryptophan biosynthesis. Initiation of transcription of the trp operon is regulated by a repressor protein that functions similarly to the lac repressor. The main difference is that the trp repressor action is subject to control by the small-molecule effector, tryptophan. When tryptophan binds the repressor, the repressor binds to the trp operator. Thus, the effect of the small-molecule effector here is opposite to its effect on the lac operon. When tryptophan is present, there is no need for the enzymes that synthesize tryptophan. 

The trp operon has a control locus called an attenuator about 150 bases after the transcription initiation site. The attenuator is regulated by the level of charged tryptophan tRNA, so that between 10% and 90% of the elongating RNA polymerases transcribe through this site to the end of the operon. Low levels of trp tRNA encourage transcription through the attenuator. 

The trp operon contains five structural genes encoding enzymes for tryptophan biosynthesis, a trp promoter (Ptrp) and a trp operator sequence (Otrp). The operon is transcribed only when tryptophan is scarce. 

When tryptophan is lacking, a trp repressor protein (encoded by the trpR operon) is synthesized. The trp repressor dimer is inactive, cannot bind to the trp operator and so the trp operon is transcribed to produce the enzymes that then synthesize tryptophan for the cell. When tryptophan is present, tryptophan synthesis is not needed. In this situation, acting as a corepressor, tryptophan binds to the repressor and activates it so that the repressor now binds to the trp operator and stops transcription of the trp operon. 

Fig. 1. Regulation of the trp operon (a) transcription in the absence of tryptophan (b) no transcription in the presence of tryptophan.

Fig. 2. Attenuation of the trp operon. (a) When tryptophan is plentiful, sequences 3 and 4 base-pair to form a 3 4 structure that stops transcription (b) when tryptophan is in short supply, the ribosome stalls at the trp codons in sequence 1, leaving sequence 2 available to interact with sequence 3. Thus a 3 4 transcription terminator structure cannot form and transcription continues.

The function of the leader sequence is to fine tune expression of the trp operon based on the availability of tryptophan inside the cell. It does this as follows. The leader sequence contains four regions (Fig. 2, numbered 1-4) that can form a variety of base-paired stem-loop ( hairpin ) secondary structures. Now consider the two extreme situations the presence or absence of tryptophan. Attenuation depends on the fact that, in bacteria, ribosomes attach to mRNA as it is being synthesized and so translation starts even before transcription of the whole mRNA is complete. When tryptophan is abundant (Fig. 2a), ribosomes bind to the trp polycistronic mRNA that is being transcribed and begin to translate the leader sequence. Now, the two trp codons for the leader peptide lie within sequence 1, and the translational Stop codon (see Topic HI) lies between sequence 1 and 2. During translation, the ribosomes follow very closely behind the RNA polymerase and synthesize the leader peptide, with translation stopping eventually between sequences 1 and 2. At this point, the position of the ribosome prevents sequence 2 from interacting with sequence 3. Instead sequence 3 base-pairs with sequence 4 to form a 3 4 stem loop which acts as a transcription terminator. Therefore, when tryptophan is present, further transcription of the trp operon is prevented. If, however, tryptophan is in short supply (Fig. 2b), the ribosome will pause at the two trp codons contained within sequence 1. This leaves sequence 2 free to base pair with sequence 3 to form a 2 3 structure (also called the anti-terminator),... 

The trp operon has no positive control system like cAMP-CAP, but it does have an additional transcriptional control mechanism that depends on the concentration of tryptophan. This involves the attenuator site, which resides within the leader (L) sequence. It consists of 14 adjacent codons beginning with a methionine codon (AUG) and ending with a termination codon (UGA) and, importantly, codons (UGG) for tryptophan at positions 10 and 11. When tryptophan is plentiful, the complete 14-residue polypeptide (leader polypeptide) is synthesized. When tryptophan is scarce, the ribosome stalls at the tandem UGG... 

TrpR, which is a DNA binding repressor protein, regulates transcription initiation of the E. coli trpEDCBA operon. Under tryptophan limiting conditions, TrpR represses transcription initiation, whereas repression is relieved in the presence of excess tryptophan. Once transcription initiates the elongating transcription complex is subject to control by transcription attenuation (reviewed in References 5 and 6). The leader transcript can form three RNA secondary structures that are referred to as the pause hairpin, the antiterminator structure, and an intrinsic terminator hairpin. Because the antiterminator shares nucleotides in common with the terminator, their formation is mutually exclusive. The pause hairpin has two additional roles in this transcription attenuation mechanism it serves as an anti-antiterminator stmc-ture that prevents antiterminator formation, and it codes for a leader peptide. A model of the E. coli trp operon transcription attenuation mechanism is presented in Fig. 2a. 

The proteins encoded by the trp operon are involved in the synthesis of tryptophan. 

---