<P> Attenuation is a second mechanism of negative feedback in the trp operon . The repression system targets the intracellular trp concentration whereas the attenuation responds to the concentration of charged tRNA . Thus, the trpR repressor decreases gene expression by altering the initiation of transcription, while attenuation does so by altering the process of transcription that's already in progress . While the TrpR repressor decreases transcription by a factor of 70, attenuation can further decrease it by a factor of 10, thus allowing accumulated repression of about 700-fold . Attenuation is made possible by the fact that in prokaryotes (which have no nucleus), the ribosomes begin translating the mRNA while RNA polymerase is still transcribing the DNA sequence . This allows the process of translation to affect transcription of the operon directly . </P> <P> At the beginning of the transcribed genes of the trp operon is a sequence of at least 130 nucleotides termed the leader transcript (trpL). Lee and Yanofsky (1977) found that the attenuation efficiency is correlated with the stability of a secondary structure embedded in trpL, and the 2 constituent hairpins of the terminator structure were later elucidated by Oxender et al. (1979). This transcript includes four short sequences designated 1--4, each of which is partially complementary to the next one . Thus, three distinct secondary structures (hairpins) can form: 1--2, 2--3 or 3--4 . The hybridization of sequences 1 and 2 to form the 1--2 structure is rare because the RNA polymerase waits for a ribosome to attach before continuing transcription past sequence 1, however if the 1--2 hairpin were to form it would prevent the formation of the 2--3 structure (but not 3--4). The formation of a hairpin loop between sequences 2--3 prevents the formation of hairpin loops between both 1--2 and 3--4 . The 3--4 structure is a transcription termination sequence (abundant in G / C and immediately followed by several uracil residues), once it forms RNA polymerase will disassociate from the DNA and transcription of the structural genes of the operon cannot occur (see below for a more detailed explanation). The functional importance of the 2nd hairpin for the transcriptional termination is illustrated by the reduced transcription termination frequency observed in experiments destabilizing the central G + C pairing of this hairpin . </P> <P> Part of the leader transcript codes for a short polypeptide of 14 amino acids, termed the leader peptide . This peptide contains two adjacent tryptophan residues, which is unusual, since tryptophan is a fairly uncommon amino acid (about one in a hundred residues in a typical E. coli protein is tryptophan). The strand 1 in trpL encompasses the region encoding the trailing residues of the leader peptide: Trp, Trp, Arg, Thr, Ser; conservation is observed in these 5 codons whereas mutating the upstream codons do not alter the operon expression . If the ribosome attempts to translate this peptide while tryptophan levels in the cell are low, it will stall at either of the two trp codons . While it is stalled, the ribosome physically shields sequence 1 of the transcript, preventing the formation of the 1--2 secondary structure . Sequence 2 is then free to hybridize with sequence 3 to form the 2--3 structure, which then prevents the formation of the 3--4 termination hairpin, which is why the 2--3 structure is called an anti-termination hairpin . In the presence of the 2--3 structure, RNA polymerase is free to continue transcribing the operon . Mutational analysis and studies involving complementary oligonucleotides demonstrate that the stability of the 2--3 structure corresponds to the operon expression level . If tryptophan levels in the cell are high, the ribosome will translate the entire leader peptide without interruption and will only stall during translation termination at the stop codon . At this point the ribosome physically shields both sequences 1 and 2 . Sequences 3 and 4 are thus free to form the 3--4 structure which terminates transcription . This terminator structure forms when no ribosome stalls in the vicinity of the Trp tandem (i.e. Trp or Arg codon): either the leader peptide is not translated or the translation proceeds smoothly along the strand 1 with abundant charged tRNAtrp . More over, the ribosome is proposed to only block about 10 nts downstream, thus ribosome stalling in either the upstream Gly or further downstream Thr do not seem to affect the formation of the termination hairpin . The end result is that the operon will be transcribed only when tryptophan is unavailable for the ribosome, while the trpL transcript is constitutively expressed . </P> <P> This attenuation mechanism is experimentally supported . First, the translation of the leader peptide and ribosomal stalling are directly evidenced to be necessary for inhibiting the transcription termination . Moreover, mutational analysis destabilizing or disrupting the base - pairing of the antiterminator hairpin results in increased termination of several folds; consistent with the attenuation model, this mutation fails to relieve attenuation even with starved Trp . In contrast, complementary oligonucleotides targeting strand 1 increases the operon expression by promoting the antiterminator formation . Furthermore, in histidine operon, compensatory mutation shows that the pairing ability of strands 2--3 matters more than their primary sequence in inhibiting attenuation . </P>

What does tryptophan bind to when it is present in e coli