Tryptophan operon mRNA

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]. 

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 ... 

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... 

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. 

It seems likely that regulating factors such as the trpR repressor products of E. coli and S. typhimurium are very similar. Somerville [200] introduced an episome-borne E. coli tryptophan operon into an S. typhimurium strain from which the tryptophan operon had been deleted. The E. coli tryptophan operon in the S. typhimurium cytoplasm was regulated in a manner essentially identical to that observed in E. coli cells. This is consistent with reports indicating a close evolutionary relationship among the tryptophan operons of the Enterobacteriaceae. Balbinder [201] found that the TS-a and TS- subunits from different species of enterobacteria interact with each other to give the activity of the complete TS enzyme. DiCamelli and Balbinder [202] studied the association of the subunits from E. coli and S. typhimurium. Denney and Yanofsky [203] reported that tryptophan mRNA from various Enterobacteriaceae species, including S. typhimurium, hybridize with E. coli tryptophan operon DNA, although not as well as E. coli tryptophan mRNA. 

The tryptophan operon of E. coli is transeribed sequentially from the trpE to the trpA gene end [130,139,161,224] into an mRNA molecule of about 6700 nucleotides [225]. Repression by tryptophan blocks the... 

Morse, D. E. Delayed-early mRNA for the tryptophan operon An effect of chloramphenicol. Cold Spr. Harb. Symp. quant. Biol. 35, 495-496 (1970). 

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... 

Regulatory sequence 1 is crucial for a tryptophan-sensitive mechanism that determines whether sequence 3 pairs with sequence 2 (allowing transcription to continue) or with sequence 4 (attenuating transcription). Formation of the attenuator stem-and-loop structure depends on events that occur during translation of regulatory sequence 1, which encodes a leader peptide (so called because it is encoded by the leader region of the mRNA) of 14 amino acids, two of which are Trp residues. The leader peptide has no other known cellular function its synthesis is simply an operon regulatory device. 

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),... 

How does the level of tryptophan alter transcription of the trp operon An important clue was the finding that the 14-amino-acid leader peptide includes two adjacent tryptophan residues. A ribosome is able to translate the leader region of the mRNA product only in the presence of adequate concentrations of tryptophan. When enough tryptophan is present, a stem-loop structure... 

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. 

A second level of control of the tryptophan biosynthetic pathway was discovered by Charles Yanofsky when he characterized mutants in the trp operon that did not affect Trp repressor binding. Yanofsky and his colleagues characterized a novel form of transcriptional control they called attenuation, which depends on the unique linkage between transcription and translation in prokaryotes. As shown in Figure 28.11, the intracellular concentration of TRP-tRNATrp determines if the ribosome will pause at a set of codons in the trp mRNA that specify consecutive Trp residues. When tryptophan levels are high, and TRP-tRNATrp is available, then the transcriptional termination hairpin loop forms and RNA polymerase disengages from the DNA template just downstream of a polyuridine... 

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