Other Attenuated Systems: Operons, Bacillus subtilis and HIV

Other Attenuated Systems: Operons, Bacillus subtilis and HIV

Study of the sequences of a number of amino acid biosynthetic operons has revealed that they are likely to be regulated by attenuation. Their leader peptides contain dramatic runs of the amino acid whose synthe-sis the operon codes for (Fig. 13.15). In summary, the attenuation mechanism seems to be an exceptionally efficient method regulating the amino acid biosynthetic operons because the necessary regulation is

his  met - thr - arg - val - gln - phe - lys - HIS - HIS - HIS - HIS - HIS - HIS - HIS - pro

ile met - thr - ala - LEU - LEU - arg - VAL - ILE - ser - LEU - VAL - ILE - ser - VAL - VAL pro - pro - cys - gly - ala - ala - leu - gly - arg - gly - lys - ala

 leu met - ser - his - ile - val - arg - phe - thr - gly - LEU - LEU - LEU - LEU - asn - ala gly - arg - pro - val - gly - gly - ile - gln - his

phe  met - lys - his - ile - pro - PHE - PHE - PHE - ala - PHE - PHE - PHE - thr - PHE - pro

thr met - lys - arg - ILE - ser - THR - THR - ILE - THR - THR - THR - ILE - THR ala - gly

trp  met - lys - ala - ile - phe - val - leu - lys - gly - TRP - TRP - arg - thr - ser

Figure 13.15 Sequences of leader peptides from some amino acid operons.

obtained by the properties of just 160 nucleotides of RNA. In the case of the trp operon, the double-barreled regulation provided by trp re-pressor and attenuation provides up to a 700-fold regulation range, 70-fold coming from repression and another 10-fold from attenuation. The autoregulation of trpR permits rapid accumulation of optimal enzyme level in cells on tryptophan starvation followed by a slower rate of enzyme synthesis when steady-state conditions have been reached.

It is not just amino acid biosynthetic operons that are regulated by attenuation. The synthesis of aspartic transcarbamoylase is also regu-lated by such a mechanism. This enzyme ultimately leads to the synthe-sis of uracil and hence UTP, and therefore we can imagine a coupling between UTP levels and the speed of transcription in a leader region, and indeed such has been found.

Escherichia coli and a number of closely related bacteria regulate their trp operons similarly. In Bacillus subtilis however, a less closely related bacterium, has evolved a significant variation of the attenuation mechanism. Introduction of multiple copies of trp operon DNA into B. subtilis leads to the deregulation of the chromosomal gene copy. Thisbecomes active whether or not tryptophan is present. One might first guess that the multiple gene copies merely bind a limited number of repressor molecules so that overall, any copy of the operon is derepressed because on average it is repressed only a small part of the time.

The actual situation on the trp operon was more interesting than mere repressor titration. Expressing trp messenger under control of the lac promoter provided a simple demonstration that it is multiple copies of trp messenger RNA and not multiple copies of DNA that lead to deregu-lation. While the lac promoter is repressed and little trp messenger is present, the cellular trp promoter is regulated normally. When trp messenger is synthesized at high rates under control of the lac promoter, the chromosomal trp operon loses regulation and becomes constitutive. The simplest explanation of this finding is that the trp RNA itself sequesters a molecule present in the cell in limited amounts. This molecule could be required for repression of the trp operon.

Simply by deleting portions of the trp messenger, the region necessary for titration of the presumed protein could be mapped. Not surprisingly, it lies ahead of the trp structural genes. The messenger ahead of the genes also has the potential to form multiple hairpin structures. One of the structures contains the typical transcription termination signal of a G-C rich hairpin followed by a string of U’s. The alternative structure to the RNA precludes formation of the termination hairpin. In the presence of tryptophan the regulatory protein binds to the structure that leads to termination at the attenuator.

The human immunodeficiency virus HIV-1 regulates synthesis of its RNA via an attenuation mechanism. Of course, since the transcription occurs in the nucleus, it cannot be directly coupled to translation as it is for the trp operon. In the absence of the HIV-1 Tat protein, RNA polymerase begins transcription at the HIV promoter, but pauses after synthesis of about 60 nucleotides. These transcripts usually terminate. When Tat protein is provided, the transcripts elongate to completion. Although Tat may interact with the promoter to affect its activity as well, the primary target of Tat action is the elongating mRNA, and Tat binds to a site on this RNA called TAR.