All the regulatory mechanisms so far control the initiation of transcription. Prokaryotes also employ some regulatory mechanisms that operate after the initiation step. A classic example was discovered when Charles Yanofsky and his colleagues found that the trp operon of E coli has a novel type of regulatory site located between the promoter/operator and the operon’s first gene, trpE. This stretch of DNA, called the leader sequence (or I), is transcribed to produce a leader mRNA segment, 162 nucleotides long, located at the end of the polycistronic trp mRNA.

Analysis of trp operson transcripts made under various conditions revealed that, as expected, the full-length, polycistronic trp mRNA is transcribed when tryptophan is scarce. This allows the enzymes of the tryptophan bio-synthetic pathway to be synthesised, and hence the pathway can produce more tryptophan. On the other hand, when tryptophan is plentiful, the genes coding for the enzymes of the tryptophan pathway are not transcribed, also as expected. An unexpected result, however, was that the DNA corresponding to most of the leader sequence is transcribed under such conditions. Based on these findings, Yanofsky suggested that the leader sequence contains a control region that is sensitive to tryptophan levels. This control sequence somehow determines not whether trp operon transcription can begin, but whether it will continue to completion. The effect of this control element was called attenuation because of its role in attenuating, or reducing, the synthesis of mRNA.

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How the attenuation mechanism works, it must start with a closer look at the trp operon leader mRNA segment. This leader has two unusual features that enable it to play a regulatory role. First, in contrast to the non-translated leader sequences typically encountered at the end of mRNA molecules, a portion of the trp leader sequence is translated, forming a leader peptide 14 amino acids long. Within the mRNA sequence coding for this peptide are two adjacent codons for the amino acid tryptophan; these will prove important. Second, the trp leader mRNA also contains four segments (labelled regions 1, 2, 3, and 4) whose nucleotides can base-pair with each other to form several distinctive hairpin loop structures. The region comprising regions 3 and 4 plus an adjacent string of eight U nucleotides is called the terminator. When base pairing between regions 3 and 4 creates a hairpin loop, it acts as a transcription termination signal.

As Yanofsky’s experiments suggested, translation of the leader RNA plays a crucial role in the attenuation mechanism. A ribosome attaches to its first binding site on the trp mRNA as soon as the site appears, and from there it follows close behind the RNA polymerase. When tryptophan levels are low, the concentration of tryptophanyl tRNA (tRNA molecules carrying tryptophan) is also low. Thus, when the ribosome arrives at the tryptophan codons of the leader RNA, it stalls briefly, awaiting the arrival of tryptophanyl tRNA. The stalled ribosome blocks region 1, allowing an alternative hairpin structure to form by pairing regions 2 and 3. When region 3 is tied up in this way, it cannot pair with region 4 to create a termination structure, and so the RNA polymerase continues, eventually producing a complete mRNA transcript of the trp operon. Ribosomes use this mRNA to synthesise the tryptophan pathway enzymes, and production of tryptophan therefore increases.

The writer is Associate Professor, Head, Department Of Botany, Ananda Mohan College, Kolkata, and also Fellow, Botanical Society of Bengal, and can be contacted at tapanmaitra59@yahoo.co.in