Jacob and Monod's Operon Model of gene expression is, fundamentally, one that invokes negative regulation of expression. In other words, it postulates that regulatory proteins are required to prevent expression.
In the case of the lac operon, the bacterial cell does not need lactose metabolizing proteins expressed if there is no lactose present to act as a substrate. It makes sense, therefore, that the regulatory protein - the repressor - blocks transcription unless lactose is present.
What if the cell needs to synthesize rather than catabolize? Can the same operon model be used to explain regulation of expression?
Yes, it can! The following diagram shows how:
In this situation, the repressor is unable to bind to the operator by itself. Hence, structural genes will be expressed at a level that is determined by the strength of the promoter.
When, the supply of a molecule is sufficient, or when it builds up to sufficient levels, it can bind to the repressor, alter its conformation, and render it unable to bind to its operator. A molecule that acts in this way is called an effector.
The Tryptophan Operon
The trp operon is an example of a biosynthetic operon whose expression is regulated by an effector:
The operon consists of 5 structural genes that code for the three enzymes required to convert chorismic acid into tryptophan:
- Anthranilate synthetase
component I - encoded by trpE
component II - encoded by trpD
- N-(5'-phosphoribosyl)-anthranilate isomerase/Indole-3-glycerol phosphate synthase - encoded by trpC
- Tryptophan synthase
b-subunit - encoded by trpB
a-subunit - encoded by trpAThe operon also contains a gene coding for a short oligopeptide (trpL) which functions in attenuation (below).
Expression is regulated by the Trp repressor protein which is encoded by the trpR gene. Unlike the lac operon, trpR is not adjacent to the operon. The structural genes are located at minute 28 on the E. coli map; trpR is located at minute 100. The TrpR repressor actually regulates the expression of two other operons in addition to the trp operon.
TrpR protein is unable to bind to the operator by itself. As long as there is insufficient tryptophan in the cell, it will remain so. However, once the level of tryptophan builds up, then the Trp repressor will block further transcription of the operon and, as a result, the synthesis of the three enzymes will decline.
View The E. coli trp Repressor Chime Movie! by Cathy Lawson. This movie illustrates the structural differences between crystal structures of the inactive apo form (3WRP) and liganded active form (1TRO) of trp repressor.
Attenuation
A straightforward prediction of the operon model of gene expression applied to the case of the trp operon is that expression of the operon in trpR mutants should be insensitive to added tryptophan. If there is no represssor to affect transcription, then since it is repressor that senses the presence of tryptophan, there should be no effect.
But there was! Expression of trpEDCBA is reduced by the addition of trypophan in trpR mutants.
Further research established that this second level of tryptophan control involved two components:
- tRNA, specifically tryptophanyl-tRNATrp, i.e. tRNATrp charged with tryptophan.
- the trpL gene
trpL codes for a short 14 aa oligopeptide. It contains two consecutive trp codons and therefore serves as a measure of the tryptophan supply in the cell. If the supply is good, then the tRNA will be charged and the leader peptide will be translated without problem. If the supply is inadequate, then the tRNA will not be charged, and translation will stall at the trp codons.
How does this affect transcription of the trp operon?
The explanation derives from the observation that the trpL mRNA region can adopt a number of different conformations. It contains several self-complementary regions which can form a variety of stem-loop structures as shown:
In one conformation, shown at the left, a typical bacterial transcription terminator can form as a result of pairing between regions 3 (green box) & 4 (yellow box). In the other conformation, at right, the terminator is precluded from forming because region 3 is now paired with region 2 (purple box) forming an alternative structure.
Now, what governs the formation of the terminator or the anti-terminator?
Recall that transcription and translation can occur simultaneously in bacteria. This means that the ribosome will attach to mRNA and is able to influence the formation of secondary structures by the mRNA.
In the case of the trpL mRNA, if there is a plentiful supply of tryptophan, then the ribosome follows right behind RNA polymerase until it is halted by a stop codon. This permits formation of the terminator stem-loop which will cause RNA polymerase to dissociate.
Notice the UGA stop codon at the bottom of region 1 in the sequence diagram above.
If the supply of tryptophan is low, then tRNATrp will not be charged with tryptophan, and the ribosome will stall waiting for a suitable tRNA to be brought to the A site:
Because the ribosome stalls, region 2 (purple) can now base-pair with region 3 (green) as soon as both are transcribed. Formation of the anti-terminator prevents formation of the terminator.
Attenuation in other operons
Attenuation as a means of regulating expression occurs in a number of other amino acid biosynthetic operons. In all cases, the leader region is rich in codons for the particular amino acids that are synthesized by the enzymes encoded by the particular operon and it can form two alternative stem-loop structures, one of which is a transcription terminator.
The following table shows the amino acid sequences of some leader peptides:
Operon Leader
LengthSequence trp 14 MKAIFVLKGWWRTS pheA 16 MKHIPFFFAFFFTFP his 16 MTRVQFKHHHHHHHPD thr 21 MKRISTTITTTITITTQNGAG leu 28 MSHIVRFTGLLLLNAFIVRGRPVGGIQH ilv 32 MTALLRVISLVVISVVVIIIPPCGAALGRGKA NOTE: (1) The number of sensing codons reflects the abundance of tRNAs for those amino acids in the cell. (2) The thr and ilv operons code for enzymes that are required in the biosynthesis of more than one amino-acid as indicated
