Trp operon

Question 1

Tryptophan operon: repressible operon

Answer 1

Codes for enzymes which synthesize amino acid tryptophan

It is regulated by repression and attenuation

Question 2

Repression

Answer 2

Controls the initiation of transcription

Question 3

Attenuation

Answer 3

Governs the frequency of premature transcript termination

Question 4

Repressor protein

Answer 4

Can be activated by allosteric transition

Question 5

TrpEDCBA genes

Answer 5

5 structural genes that code for enzymes that convert chorismic acid to tryptophan

Transcribed as a polycistronic mRNA that directs translation of enzymes to catalyze the biosynthesis of tryptophan

Question 6

Pathway of chorismic acid to tryptophan

Answer 6

CA -> anthranilate -> phosphoribosyl anthranilate -> indole-3-glycerol phosphate -> tryptophan

Question 7

trpP

Answer 7

Promoter region

Question 8

trpO

Answer 8

Operator region

Question 9

trpR gene

Answer 9

Located away from the tryptophan structural genes

Codes for trp operator protein

Question 10

repression

Answer 10

controls the initiation of transcription

Question 11

When tryptophan is absent

Answer 11

Trp operon is activated.

trpR is a repressor protein

Trp repressor is an inactive protein which is unable to bind to trp operon on its own

RNA polymerase bind to promoter region and transcribes the trpEDCBA structural genes to produce polycistronic mRNA

The trpEDCBA polycistronic mRNA is then translated by the ribosomes into the enzymes that synthesize the amino acid tryptophan.

Question 12

When tryptophan is present

Answer 12

trp operon is inactivated

tryptophan acts as co-repressor and binds to inactive trpR repressor protein, cause an allosteric change to repressor protein

This tryptophan-repressor complex is now able to bind to the operator region, repressing the transcription of the 5 structural genes by blocking access to RNA polymerase.

Thus when tryptophan is present in excess, the tryptophan operon is repressed and the enzymes necessary for tryptophan synthesis are not
made.

Question 13

Attenuation

Answer 13

A regulatory mechanism to prematurely terminate transcription of the mRNA

Another mechanism by which tryptophan inhibits enzyme synthesis.

The activated repressor (i.e. TrpR + tryptophan), even when bound to the operator region, does not strongly inhibit expression of the trp operon

Question 14

Leader sequence in the trp operon

Answer 14

• > mRNA transcription is initiated by RNA polymerase within the trpP region and proceeds along a leader sequence.
• > Within this leader sequence, there are regulatory regions which is an integral part of the control mechanism of the trp operon.

-> When transcribed by RNA polymerase, the leader mRNA sequence can folds back on itself and form a stem loop (hairpin) via complementary
base-pairing

Question 15

Leader sequence in trp operon

Answer 15

• > ribosomal binding site

- > a mini open reading frame

Question 16

The mini ORF in leader sequence

Answer 16

• > AUG translation start codon
• > 2 trp (UGG) codons responsible for attenuation
• > UGA translation stop codon

Question 17

Molecular explanation for attenuation

Answer 17

• Translation of the trpL gene produces a short polypeptide.
• Near the stop codon are two tryptophan (UGG) codons.
• Within the leader mRNA sequence are four regions that can form secondary structures by complementary base-pairing.
• Tight coupling of transcription and translation in prokaryotes makes control by attenuation possible.
• During the pause, a ribosome loads onto the mRNA and begins translation of the leader peptide.
• Ribosome position is key to attenuation.
• Secondary structure of the leader mRNA sequence leads to either continued transcription or termination (attenuation) by the RNA polymerase.

Question 18

2 important stem loop hairpin structures:

Answer 18

Hairpin 3+4 -> termination hairpin (in presence of tryptophan)

Hairpin 2+3 -> anti-terminator (in absence of tryptophan)

Question 19

When tryptophan concentration is high

Answer 19

Transcription of the leader sequence proceeds but premature termination (attenuation) of the mRNA synthesis occurs. Incomplete trp operon is transcibed

• > Then charged tRNAtrp is also present.
• > The ribosome successfully translate the leader sequence, ending in region 2.
• > This prevents region 2 from pairing with region 3, leaving region 3 available to pair with region 4.

-> Pairing of regions 3 and 4 creates a rho-independent terminator known as the
attenuator.

-> The hairpin formed by regions 3+4 is a signal for RNA polymerase to terminate transcription before the structural genes are reached.

Question 20

When tryptophan is absent or low in concentration

Answer 20

The inactive Trp repressor cannot bind to the operator and attenuation is bypassed. Entire trp operon is transcribed by RNA polymerase.

• > Then charged tRNAtrp are unavailable.
• > The ribosome stalls during the translation of the trp (UGG) codons in the leader sequence, covering region 1.
• > When region 1 is covered by the ribosome, it cannot pair with region 2.
• > Region 2 pairs with region 3 instead.
• > The hairpin formed by regions 2+3 is a signal for RNA polymerase to continue mRNA transcription of the trpEDCBA structural genes.
• > Once region 3 is paired with region 2, it is unable to pair with region 4.
• > Without the hairpin formed by regions 3+4, attenuation is bypassed and transcription continues through the structural genes.

Question 21

Conclusion for trp operon

Answer 21

Thus, mRNA transcription may still occur when the Trp
repressor is bound to the operator, but transcription is
terminated in the presence of high concentration of
tryptophan through the attenuation mechanism