Question

11. Is the lac operon a repressible or an inducible operon? What happens in the absence of lactose? What happens in the presence of lactose? Is the trp operon a repressible or an inducible operon? What happens in the absence of tryptophan? What happens in the presence of tryptophan?

Answer 1

Lac operon is an inducible operon.  In the absence of lactose the repressor tetramer remains bound to the operator (lacO) and that prevents transcription initiation from the promoter of the operon (lacP). The repressor protein encoded by the lacI gene is an allosteric protein and the active (DNA binding) form of the protein is a tetramer. Each monomer in the repressor tetramer has an allosteric binding site for lactose. In the lactose bound conformation the repressor tetramer cannot bind to the operator of lac operon (lacO) and therefore RNA polymerase can initiatiate transcription from lacP.

Tryptophn operon is a repressibible operon. The trp repressor is a tetramer of four identical subunits of about 100 amino acids each. Under normal conditions about 20 molecules of the repressor tetramers are present in the cell. The repressor by itself does not bind to trpO, the operaor of the operon. For binding to the trpO the repressor tetramer binds to the effector molecule, tryptophan. This is exactly opposite to the lac repressor. The binding of repressor-co-repressor (tryptophn) complex to the trpO physically blocks the binding of RNA polymerase to the promoter trpP. So when the tryptophan concentration in the cell is sufficient, the repressor-co-repressor complex binds to the operator and prevents the transcription of tryptophan operon. When the concentration of tryptophan in the cell is very low, the repressor tetramer cannot form the repressor-co-repressor complex and it cannot bind to the operaator of tryptophan operon. Therefore RNA polymerase can initiate transcription from trpP.

Another important control element of trp operon and not present in lac operon is the attenuator site. It lies within 162 nucleotides between the start of transcription from trpP and the initiation codon of trpE gene (the first strctural gene in the transcription unit). The trpE gene starts at +163. Within this first 162 nucleotides called the leader sequence there is a coding region (fourteen codons) beginning with an initiation codon and ends in a termination codon. This coding sequence is preceded by a usual ribosome binding site and therefore translation of this coding sequence could yield a 14 amino acid long leader peptide. This peptide has never been detected in bacterial cells, perhaps it is degraded very rapidly.

The attenuator region provides RNA polymerase with a second chance to stop transcription if the trp enzymes are not needed by the cell. In the presence of tryptophan it acts like a rho independent transcription termination site to produce a short 140 nucleotide long transcript. In the absence of tryptophan, it has no effect on ranscription and a full length mRNA is made. In the presence of tryptophan, the attenuator stops transcription by those RNA polymerases that have escaped repression at trpO. The termination of transcription at the attenuator site is made possible because of the coupled nature of bacterial transcription and translation. Translating ribosomes play an active role in attenuator dependent transcription termination. Leader peptide of 14 amino acids contains two adjacent tryptophans at positions 10 and 11. This is unusual because tryptophan is a rare amino acid in E. coli. If the tryptophan level in the cell is very low, the amount of charged tRNAtrp will also be low and the ribosome may be unable to translate the leader peptide through the two trp codons. Therefore the ribosome will stall at this place in the leader RNA sequence.

RNA sequence at the leader region can adopt several possible secondary structures. The position of the ribosome within the leader peptide coding sequence determines the secondary structure that will form. This secondary structure in turn is recognized by RNA polymerase that has just transcribed through the attenuator coding region and is now located a small distance downstream. The secondary structure formed in RNA decides whether transcription should terminate or continue into trpE gene. The attenuator region can be divided into four regions based on the complementarity of sequences. Region 2 is complementary to 1 and 3 and region 3 is complementary to 2 and 4. In the presence of enough tryptophan ribosome does not stall at the two tandem tryptophan codons- UGGUGG, near the beginning of region 1 but continues translation into region 2. When the ribosome is in region 2, region 1 and 2 cannot base pair but region 3 & 4 can form base pairs resulting in a hair pin loop followed by eight U residues – a feature found in rho independent transcription terminators. This hair pin is sensed by RNA polymerase and termination of transcription occurs. Thus, when the leader RNA sequence is being synthesized in presence of sufficient tryptophan, a stem loop is formed between regions 3 & 4 and termination of transcription occurs. In the absence of tryptophan ribosome is stalled at trp codons and region 1 is prevented from base pairing with region 2. Region 2 can now base pair with region 3 so that region 4 remains single stranded. Therefore the stem loop involving regions 3 & 4 is not formed and the RNA polymerase continues on with its transcription. Thus, for transcription to proceed past the attenuator, region 1 must be prevented from base pairing with region 2. When ribosome is stalled at region 1 region 2 cannot base pair with region 1. Since the regions 2 & 3 are synthesized before region 4 by RNA polymerase they in turn will base pair before region 4 appears in the newly transcribed RNA. Therefore, region 4 remains single single stranded and RNA polymerase continues transcription into the structural genes. Under conditions in which the leader peptide is not translated, stem loop 1-2 will form preventing the formation of stem loop 2-3. This permits the formation of stem loop 3-4 which signals transcription termination.