Trp codon

When tryptophan levels are low, the ribosome pauses at the Trp codons in sequence 1. 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 2 3 structure, unlike the 3 4 attenuator, does not prevent transcription. 

When tryptophan concentrations are high, concentrations of charged tryptophan tRNA (Trp-tRNATrp) are also high. This allows translation to proceed rapidly past the two Trp codons of sequence 1 and into sequence 2, before sequence 3 is synthesized by RNA polymerase. In this situation, sequence 2 is covered by the ribosome and unavailable for pairing to sequence 3 when sequence 3 is synthesized the attenuator structure (sequences 3 and 4) forms and transcription halts (Fig. 28-21b, top). When tryptophan concentrations are low, however, the ribosome stalls at the two Trp codons in sequence 1, because charged tRNATrp is less available. Sequence 2 remains free while sequence 3 is synthesized, allowing these two sequences to base-pair and permitting transcription to proceed (Fig. 28-21b, bottom). In this way, the proportion of transcripts that are attenuated declines as tryptophan concentration declines. 

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. 

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),... 

Fig. 16.8. Attenuation of the trp operon. Sequences 2, 3, and 4 in the mRNA transcript can form base pairs (2 with 3 or 3 with 4) that generate hairpin loops. When tryptophan levels are low, the ribosome stalls at the adjacent trp codons in sequence 1, the 2-3 loop forms, and transcription continues. When tryptophan levels are high, translation is rapid and the ribosome blocks formation of the 2-3 loop. Under these conditions, the 3 loop forms and terminates transcription.

FIGURE 11.16 The attenuation mechanism in the trp operon. The pause structure forms when the ribosome passes over the Trp codons quickly when tryptophan levels are high. This causes premature abortion of the transcript as the terminator loop is allowed to form. When tryptophan is low, the ribosome stalls at the Trp codons, allowing the antiterminator loop to form, and transcription continues. 

Mutant D mutations replacing the two Trp codons "with Leu codons Mutant E mutant E with a mutation in the Leu-tRNA synthetase gene Mutant F mutant E that constitutively synthesizes leucine... 

Mutant D Removing the Trp codons would lose all regulation by tryptophan, and Trp synthesis would be regulated by the levels of leucine. In this mutation, the Trp synthesis genes would not be transcribed even in the absence of tryptophan, and this strain, like mutants B-D, would always require tryptophan to grow. 

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.

As more RNA is synthesized, transiation keeps pace with transcription. Region A has been synthesized and B is Just beginning. The ribosome is about to translate the Trp codons of the leader peptide... 

Tryptophan-aminoacyl-tRNA is scarce, so the ribosome pauses at the Trp codons. As 6 is synthesized it is not partiy covered by the ribosome. Transiation is not keeping pace with transcription. 

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