Tryptophan operon

Transcription of the following sequence of the tryptophan operon occurs in the direction indicated by the arrow. What would be the base sequence of the mRNA produced ... 

The enzyme tryptophan synthase is an a2ft tetramer (i.e., it contains two pairs of identical chains).36 In the genome, the a chain (Mr = 29 000) and the j8 chain (Mr = 44 000) are the two most distal genes of the tryptophan operon. (An operon is a set of consecutive genes that may be induced or repressed as a group. The entire set of enzymes of a biosynthetic pathway is frequently encoded in this way so that the pathway can be turned on or off as a whole—coordinate expression.) The tetrameric enzyme catalyzes the following reaction ... 

Besides having a noncovalent association of subunits as in tryptophan synthase, some enzymes are double-headed, in that they contain two distinct activities in a single polypeptide chain. A good example of this is the indole 3-glycerol phosphate-synthase-phosphoribosyl anthranilate isomerase bifunc-tional enzyme from the tryptophan operon of E. coli. The crystal structure of the complex has been solved at 2.0 A resolution.39 The two enzymes have been separated by genetic manipulation.40 The activity of the two separate monomeric monofunctional constituents is the same as in the covalent complex so there is no catalytic advantage of having the proteins fused. 

The tryptophan operon, indicating the location of the different genes, the polypeptide chains, the resulting enzyme complexes, and the reactions catalyzed by the enzyme complexes. 

The leader region for the tryptophan operon. The region of the leader RNA containing the hypothesized leader polypeptide is shown. The translation start of the trpE protein is also shown. 

Figure 5.26. Complementarity between mRNA and DNA. The base sequence of mRNA (red) is the complement of that of the DNA template strand (blue). The sequence shown here is from the tryptophan operon, a segment of DNA containing the genes for five enzymes that catalyze the synthesis of tryptophan. The other strand of DNA (black) is called the coding strand because it has the same sequence as the RNA transcript except for thymine (T) in place of uracil (U).

A novel mechanism for regulating transcription in bacteria was discovered by Charles Yanofsky and his colleagues as a result of their studies of the tryptophan operon. The 7-kb mRNA transcript from this operon encodes five enzymes that convert chorismate into tryptophan (Section 24.2.10). The mode of regulation of this operon is called attenuation, and it... 

III The isolated tryptophan operon was attached to a more efficient promoter prior to integration into chromosomal DNA... 

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

Platt, T., Regulation of gene expression in the tryptophan operon of Escherichia coli, in The Operon, Miller, J. H. and Reznikoff, W. S., Eds., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1980, 2, 263-302. 

Another example of translational control in eukaryotes is the inhibition of yeast GCN4 protein synthesis by stem-loop structures present in the 50 end of the mRNA. GCN4 control, and an analogous situation in bacteria, links amino-acid biosynthesis to ribosome pausing in the 50 end of the mRNA. This mechanism was first described for the tryptophan operon in E. coli and it is often referred to as attenuation. Transcriptional and translational control of the tryptophan biosynthetic enzymes are described in Chapter 28. 

Sen, A.K. W. Liu. 1990. Dynamic analysis of genetic control and regulation of amino acid synthesis The tryptophan operon in Escherichia coli. Biotechnol. Bioeng. 35 185-94. 

Fig. 9-18 The tryptophan operon and its reguiation showing the situation with moderateiy iow concentrations of tryptophan. The terminator structure forms when the ribosome continues transiation past the trp codons in the ieader peptide and biocks region 2. With criti-caiiy iow concentrations of tryptophan, the ribosome staiis at trp codons, favoring formation of a stem ioop structure that permits transcription to continue.

Miozzari GF, Yanofsky C (1978) Translation of the leader region of the Escherichia coli tryptophan operon. J Bacteriol 133 1457-1466... 

Figure 5 Determination of reai-time PCR efficiencies from the slopes of the calibration cunre (method A), according to the equation of reference gene glutathione transferase (Gst), and two target genes tryptophan operon (TyrA) and aspartate transcarbamylase (PyrB). CP cycles versus cDNA concentration input (log scale) were plotted to calculate the slope (mean + SD n = 3),...

Considerations of initiation of lac messenger RNA synthesis are directly related to the problems of induction (see Section IV). Since within 90 seconds of induction, enzyme appears, initiation of messenger synthesis must be rapid. The relative abundance of various messengers in the cell may be a function of the rate of initiation of RNA synthesis. For different RNA s the initiation rates vary by a wide margin. Thus initiation rates for ribosomal and tRNA synthesis in rapidly growing E. coli at 30°C range from 0.5 to 1 initiation per second [36], as contrasted with an initiation rate for the tryptophan operon mRNA of 1 every 2 to 5 minutes [37,38]. 

The current concept of a classic operon is largely derived from studies of the lac operon of E. coli and includes a promoter at the beginning which is considered to be the site at which the DNA-dependent RNA polymerase initiates transcription [151,152]. The promoter is followed by a closely associated operator serving as the site of attachment for repressor molecules which can prevent or impede transcription [153]. The structural genes are next in the sequence, and in the case of the tryptophan operon they code for the peptides which make up the tryptophan biosynthetic enzymes. Presumably there is an element at the end of the operon which acts as a signal to terminate transcription. This section will describe information derived primarily from work with E. coli and S. typhimurium. A number of general reviews and discussions of regulation have been published recently [4-6,105,154]. 

As described in a previous section, five structural genes have been identified in the tryptophan operons of E. coli and S. typhimurium (Fig. 3). At one end of the cluster is the promoter, PI, and the operator, which serve, respectively, as the site for initiation of transcription and as a target for the repressor and together largely determine the level of expression of the structural genes. As mentioned in the section on regulation of enzyme synthesis, there is evidence for an internal promoter-like... 

Fig. 4. Diagram of the tryptophan operon plus two neighboring genes. The gene designations for both E. coli and S. typhimurium are given. The supX locus has not been identified in E. coli. Controlling elements PI, the promoter associated with the operator 0, the operator P2, the low-efficiency internal promoter. Enzymes AS-I, anthianilate synthetase component I PRT, phosphoribosyl transferase (AS-II, anthianilate synthetase component II) PRAI, phosphoribosyl anthianilate isomerase InGPS, indoleglycerol phosphate synthetase TS-a, tryptophan synthetase a-chain TS-)S, tryptophan synthetase j8-chain. RUM, region of unusual mutations in S. typhimurium.

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