TRYPTOPHAN OPERON Bacteria such as E. coli need amino acids to survive. Tryptophan is one such amino acid that E. coli can ingest from the environment. E. coli can also synthesize tryptophan using enzymes that are encoded by five genes. These five genes are next to each other in what is called the tryptophan (trp) operon. If tryptophan is present in the environment, then E. coli does not need to synthesize it; the switch controlling the activation of the genes in the trp operon is turned off. However, when tryptophan availability is low, the switch controlling the operon is turned on, transcription is initiated, the genes are expressed, and tryptophan is synthesized. A DNA sequence that codes for proteins is referred to as the coding region. The five coding regions for the tryptophan biosynthesis enzymes are arranged sequentially on the chromosome in the operon. Just before the coding region is the transcriptional start site. This is the region of DNA to which RNA polymerase binds to initiate transcription. The promoter sequence is upstream of the transcriptional start site. Each operon has a sequence within or near the promoter to which proteins (activators or repressors) can bind and regulate transcription. trp Operon Gene Gene Function P/O Promoter; operator sequence is found in the promoter trp L Leader sequence; attenuator (A) sequence is found in the leader trp E Gene for anthranilate synthetase subunit1 trp D Gene for anthranilate synthetase subunit2 trp C Gene for glycerolphosphate synthetase trp B Gene for tryptophan synthetase subunit1 trp A Gene for tryptophan synthetase subunit2 Page 1 A DNA sequence called the operator sequence is encoded between the promoter region and the first trp-coding gene. This operator contains the DNA code to which the repressor protein can bind. When tryptophan is present in the cell, two tryptophan molecules bind to the trp repressor, which changes shape to bind to the trp operator. 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. While the lac operon can be activated by a chemical (allolactose), the tryptophan (Trp) operon is inhibited by a chemical (tryptophan). This operon contains five structural genes: trp E, trp D, trp C, trp B, and trp A, which encode tryptophan synthetase. It also contains a repressive regulator Page 2 gene called trp R. Trp R has a promoter where RNA polymerase binds and synthesizes mRNA for a regulatory protein. The protein that is synthesized by trp R then binds to the operator which then causes the transcription to be blocked. In the lac operon, allolactose binds to the repressor protein, allowing gene transcription, while in the trp operon; tryptophan binds to the repressor protein effectively blocking gene transcription. In both situations, repression is that of RNA polymerase transcribing the genes in the operon. Also unlike the lac operon, the trp operon contains a leader peptide and an attenuator sequence which allows for graded regulation. Page 3 The attenuator mechanism of Trp operon The attenuator mechanism uses the 5’ end of the leader sequence (trpL) of the transcript. The leader is about 140 ntds long; it has four segments of complementary sequences, call 1, 2, 3 and 4. These sequences facilitate partial base pairing between 1 and 2, 2 and 3 and 3 and 4 to generate two stem loop structures. The 4th segment when pairs with the 3rd, it forms a stem loop similar the rho independent transcriptional terminator structure with terminal Us. In the presence of Trp, formation of this structure prevents the transcription initiated by the RNAP to proceed further and the transcription is terminated. So this is a fail-proof operation. Page 4 When Trp is absent, the leader sequence can still generate this chain termination stem loop structure and terminate transcription, but transcription of the full-length transcript is required for Tryptophan synthesis. In order to produce full-length transcript the terminator stem-loop formation should be prevented by change in base pairing alignments among the four blocks. Page 5 When tryptophan levels are high, the ribosome quickly translates sequence 1 (open reading frame encoding leader peptide) and blocks sequence 2 before sequence 3 is transcribed. Continued transcription leads to attenuation at the terminator-like structure formed by sequences 3 and 4. When tryptophan levels are low, the ribosome pauses at the Trp codons in sequence l. Formation of the paired structure between sequences 2 and 3 prevents attenuation because sequence 3 is no longer available to form the attenuator structure with sequence 4. The trp operon attenuation mechanism uses signals encoded in four sequences within a 162 nucleotide leader region at the 5' end of the mRNA that precedes the initiation codon of the first gene. At the end of the leader is a sequence called the attenuator, made up of sequences 3 and 4. Sequences 3 and 4 base-pair to form a G=C rich stem and loop structure followed by a series of uridylate residues, a structure that resembles a transcription terminator; transcription will halt here when this structure forms. Page 6