Coordinate control of genes in bacteria is widespread. In the preceding section, we looked at examples illustrating the regulation of pathways for the breakdown of specific sugars. In fact, most coordinated genes in bacteria are coordinated through operon mechanisms. In many pathways that synthesize essential molecules from simple inorganic building blocks, the genes that encode the enzymes are organized into operons, complete with multigenic mRNAs. Furthermore, in cases for which the sequence of catalytic activity is known, there is a remarkable congruence between the order of operon genes on the chromosome and the order in which their products act in the metabolic pathway. This congruence is strikingly illustrated by the organization of the tryptophan operon in E. coli (Figure 11-21). The tryptophan operon contains five genes (trpE, trpD, trpC, trpB, trpA) that encode enzymes that contribute to the synthesis of the amino acid tryptophan.

There are two mechanisms for regulating transcription of the tryptophan operon and some other operons functioning in amino acid biosynthesis. One provides global control of operon mRNA expression, and the other provides fine-tuned control.

The level of trp operon gene expression is governed by the level of tryptophan. When tryptophan is absent from the growth medium, trp gene expression is high; when levels of tryptophan are high, the trp operon is repressed. One mechanism for controlling the transcription of the trp operon is similar to the mechanism that we have already seen controls the lac operon: a repressor protein binds an operator, preventing the initiation of transcription. This repressor is the Trp repressor, the product of the trpR gene. The Trp repressor binds tryptophan when adequate levels of the amino acid are present, and only after binding tryptophan will the Trp repressor bind to the operator and switch off transcription of the operon. This simple mechanism ensures that the cell does not waste energy producing tryptophan when the amino acid is sufficiently abundant. E. coli strains with mutations in trpR continue to express the trp mRNA and thus continue to produce tryptophan when the amino acid is abundant.

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In studying these trpR mutant strains, Charles Yanofsky discovered that, when tryptophan was removed from the medium, the production of trp mRNA further increased several-fold. This finding was evidence that, in addition to the Trp repressor, a second control mechanism existed to negatively regulate transcription. This mechanism is called attenuation because mRNA production is normally attenuated, meaning “decreased,” when tryptophan is plentiful. Unlike the other bacterial control mechanisms described thus far, attenuation acts at a step after transcription initiation.

The mechanisms governing attenuation were discovered by identifying mutations that reduced or abolished attenuation. Strains with these mutations produce trp mRNA at maximal levels even in the presence of tryptophan. Yanofsky mapped the mutations to a region between the trp operator and the trpE gene; this region, termed the leader sequence, is at the 5′ end of the trp operon mRNA before the first codon of the trpE gene (Figure 11-22). The trp leader sequence is unusually long for a prokaryotic mRNA, 160 bases, and detailed analyses have revealed how a part of this sequence works as an attenuator that governs the further transcription of trp mRNA.

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The key observations are that, in the absence of the TrpR repressor protein, the presence of tryptophan halts transcription after the first 140 bases or so, whereas, in the absence of tryptophan, transcription of the operon continues. The mechanism for terminating or continuing transcription consists of two key elements. First, the trp mRNA leader sequence encodes a short, 14-amino-acid peptide that includes two adjacent tryptophan codons. Tryptophan is one of the least abundant amino acids in proteins, and it is encoded by a single codon. This pair of tryptophan codons is therefore an unusual feature. Second, parts of the trp mRNA leader form stem-and-loop structures that are able to alternate between two conformations. One of these conformations favors the termination of transcription (Figure 11-23a).

The regulatory logic of the operon pivots on the abundance of tryptophan. When tryptophan is abundant, there is a sufficient supply of aminoacyl-tRNA^Trp to allow translation of the 14-amino-acid peptide. Recall that transcription and translation in bacteria are coupled; so ribosomes can engage mRNA transcripts and initiate translation before transcription is complete. The engagement of the ribosome alters trp mRNA conformation to the form that favors termination of transcription (where segments 3 and 4 form base pairs; Figure 11-23b). However, when tryptophan is scarce, the ribosome is stalled at the tryptophan codons, segments 2 and 3 base-pair, and transcription is able to continue (Figure 11-23c).

Other operons for enzymes in biosynthetic pathways have similar attenuation controls. One signature of amino acid biosynthesis operons is the presence of multiple codons for the amino acid being synthesized in a separate peptide encoded by the 5′ leader sequence. For instance, the phe operon has seven phenylalanine codons in a leader peptide and the his operon has seven tandem histidine codons in its leader peptide (Figure 11-24).

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