Control of the trp operon by attenuation Repression and derepression can change the level of expression of structural genes

the trp operon by about 70-fold. There is a second level of regulation of trp operon expression, however. In trpR mutantd that cannot make repressor, the addition of tryptophan to a culture of cells growing in the absence of tryptophan will cause an 8 to 1-fold decrease in the rates of synthesis of the tryptophan biosynthetic enzymes. Moreover, deletions that remove part of the trpL region result in increased rates of these deletions are indeoendent of repression; the increase occurs in both the repressed and the derepressed state. This second level of regulation of the trp operon is called attenuation, and the sequence within trpL that controls this phenomenon is called the attenuator. Attenuation occurs by control of the termination of transcription at a site near the end of the mRNA leader sequence. This premature termination of trp operon transcription occurs only in the presence tryptophan-charged tRNA trf and yields a 140-nucleotide-long leader-sequence transcript. The attenuator region has a nucleotide-pair sequence essentially identical to the transcription-termination signals found at the ends of most bacterial operons. These termination signals contain a GC-rich palindrome followed by several AT base-pairs. Transcription of these termination signals yields a nascent RNA with the potensial to from a hydrogen-bonded hairpin structure followed by several Us. When a nascent transcript forms this hairpin structure, it is believed to cause a conformational change in the associated RNA polymerase, resulting in termination of transcription within the following, more weakly hydrogen-bonded region of DNA-RNA base-pairing. The nucleotide sequence of the attenuator therefore explains its ability to prematuraly terminate trp by operon transcription. But how can this be regulated by the presence or absence of tryptophan? First, recall that transcription and translation are coupled in prokaryotes, that is, ribosomes begin translating mRNAs while they are still being produced by transcriptions. Thus, events occurring during translation may also affect transcription. Second, note that the 162-nucleotide-long leader sequence of the trp operon mRNA(fig.14.10) contains sequences that can base-pair to form alternate secondary structures. Two of these sequencees form the previously mentioned transcription-termination hairpin (fig 14.11c.). this hairpin is formed by basepairing between nucleotide sequences 114-121 and 126-134 (nucleotide 1 is at the 5 terminus). An alternate secondary structure results from base -pairing between leader sequences 74-85 and 108-119 (fig. 14.11b). obviously, only one of these structures can exist at one time, since nucleotides 114-119 are part of both. Thus,

if sequences 74-85 and 108-119 are base-paired, the attenuator transcriptiontermination hairpin cannot form. Third, note that the leade, sequence contains an AUG translation-initiation codon, followed by 13 codons for amino acids, followed in turn by a UGA translationtermination codon (fig.14.10). moreover, the trp leader sequence has been shown to contain an efficient ribosome-binding site located in the appropriate position for the initiation of translation at the leader AUG initiation codon. It seems very likely that a 14-amino-acid-long leader peptide is synthesized as diagrammed in fig. 14.10. this putative leader peptide has not yet ben detected in vivo, but short peptides of this type are very rapidly degraded in E.coli, so failure to detect it is not un expected. Note that the leader peptide contains two contiguous tryptophan residues. The two Trp codons are positioned such that in the absence of trytophan (and thus the absence ot Trp-tRNA trp), the ribosome will become stalled before it encouters the base-paired structure formed by leader sequences 74-85 and 108-119 (fig. 14.11b). this base-pairing precludes the formation of the transcription-termination hairpin. Thus, in the absence pf triptophan, transcription will continue past the attenuator into the trpE gene. In the presence of tryptophan, the ribosome can translate past the trp codons to the leader-peptide termination codon. In the process, it will have to disrupt the basepairing between leader sequences 74-85 and 108-119. This , in turn, frees the 114121 sequence, allowing it to base-pair with the 126-134 sequence and form the transcription-termination hairpin (fig.14.11c). thus in the presence of tryptophan, transcription frequently terminates at the attenuator, reducing the amount of mRNA for the trp structural genes. The transcription of the trp operon can be regulated over the range of almost 700fold by the combined effect of repression (up to 70-fold) and attenuation (up to 10-fold) Regulation of transcription by attenuation is not unique to the trp operon. Six operons (trp,thr,ilv,leu,phe, and his) are known to be regulated by attenuation. Of these , trp and possibly phe are also regulated by repression. The his operon, which has long been thought to be repressible , is now believed to be regulated entirely by attenuation. Although minor details vary from operon to operon,the main features of attenuation are the same for all six operons. Feedback Inhibition And Allosteric Enzymes Earlier in this chapter, we described the mechanism by which the transcription of bacterial genes coding for enzymes in a biosynthetic pathway is repressed when the end product of the pathway is present in the medium in which the cells are

growing. A second, and more rapid, regulatory fine-tuning of metabolism often occurs at the level of enzyme activity. The presence of sufficient concentrations of an end product (such as histidine or tryptophan) of a biosynthetic pathway will frequently result in the inhibition of the first enzyme in the pathway. This phenomenonis called feedback inhibition or end product inhibution; it should not be confused with repression (inhibition of enzyme synthesis). Feedback inhibition result in an almost instantaneous arrest of the synthesis of an end product when it is added to the medium. Feedbach inhibitoin-sensitive enzymes have been shown to have an end product binding site (or sites) in shown to have an end product binding site. In the case of some multimeric enzymes, the end product or regulatory binding site is on a different subunit (polypeptide) than the subtrate site. Upon binding the end product, such enzymes are believed to undergo changes in conformation, called allosteric transitions, that reduse their affinity for their substrates. Proteins that undergo such conformational changes are usually referred to as allosteric proteins. Many example are known, including numerous feedback inhibition-sensitive enzymes and the repressor molecules discussed in the preceding sections.