attenuator sequence aug e * aug d * aug c * aug b * aug a * trp structural genes p trp o 1234...

Attenuator SequenceAUG E * AUG D * AUG C * AUG B * AUG A *

Trp structural genes

PTrp O

1 2 3 4AUG UGA UUUUUUUUUUGGUGG

The Trp operon is controlled by negative regulation. A repressor protein (Trp repressor) binds to the operator sequence (O) and prevents transcription. The repressor can only bind DNA if it is simultaneously binding a molecule of tryptophan. Thus when trp is present the trp operon is repressed by the trp repressor and turned off.

When the amount of trp present in the bacteria decreases, trp dissociates from the trp repressor and the trp repressor can no longer bind the operator and txn occurs. However, there is still some trp present (moderate amounts) so the operon does not need to be actively transcribed

Within the attenuator sequence, there are two sets of imperfect, inverted repeats. Each pair of repeats is indicated above as a colored arrow. Within repeat 1, are two trp codons (UGGUGG). Region 2 and region 3 (including dashed portion) form an inverted repeat with a single mismatch. These repeats can therefore form three possible hairpin secondary structures in the newly transcribed mRNA.

In the presence moderate amounts of trp, repeats 1 & 2 will form a hairpin, leaving repeat 3 (solid portion) and repeat 4 free to form a hairpin. Formation of the 3-4 hairpin causes termination of transcription due to the presence of the 9 U’s immediately following repeat 4. This is a classical rho-independent terminator sequence. (View Slides 2-4)

In the near absence of trp, ribosomes which bind and begin translation at the AUG just upstream of repeat 1 will pause when they reach the two adjacent trp codons within repeat 1. The presence of the ribosome at this position prevents a hairpin from forming between repeats1 and 2. Therefore, as RNA polymerase continues to transcribe the leader sequence, repeat 2 forms a hairpin structure with repeat 3 (dashed and solid portions) as soon as repeat 3 is transcribed. With repeats 2 & 3 forming a hairpin structure, repeat 4 remains single stranded and a termination hairpin is not formed. Termination does not occur and there is no attenuation of transcription. RNA polymerase continues through the remainder of the leader region and on through the structural genes E, D, C, B & A. (View Slides 5-7)

Next Slide

Trp Operon Overview

1 2 3 4AUG UGA UUUUUUUUUUGGUGG

Attenuation occurs in the presence of moderate amounts of trp

UUUUUUUUU

Ultimately, this is the secondary structure which will form if Trp is present

Next Slide

2 3 4UUUUUUUUU

1AUG UGAUGGUGG

1AUG UGAUGGUGG

4UUUUUUUUU

Attenuation is overcome when Trp levels are very low

2 3

Stalled Ribosome

Ultimately, this is the secondary structure formed at low concentrations of Trp

Next Slide

AUG UGG

RNA polymerase

Attenuation occurs at moderately low levels of Trp

The following 8 slides will play automatically to animate the process of attenuation. To begin, click the button below

Transcription of the Trp operon has begun. RNA polymerase has transcribed from the +1 into the trp leader sequence. The line and boxes represent newly synthesized mRNA from the trp operon.

Attenuation occurs at moderately low levels of Trp

A ribosome has bound to the 1st AUG of the leader sequence and begun translation. RNA polymerase has continued transcription into region 2

1 2AUG UGAUGGUGG

Ribosome

RNA polymerase

Attenuation occurs at moderately low levels of TrpAttenuation occurs at moderately low levels of Trp

Ribosome

1 2AUG UGAUGGUGG

RNA polymeraseShort peptide

Trp-tRNATrp

RNA polymerase continues transcription of the leader sequence. Translation proceeds to the two trp codons. Since there is some (moderate) amount of trp ( ) in the cell, Trp-tRNATrp is produced and can be used by the ribosome.

Attenuation occurs at moderately low levels of Trp

1 2AUG UGAUGGUGG

Ribosome

RNA polymerase

Short peptide

3

Since trp is present, translation continues through the two trp codons on to the stop codon. Transcription has continued into region 3.

Termination factor

1 2AUG UGA

3 4UGGUGG

Ribosome dissociatesafter reaching stop codon

RNA polymerase

Attenuation occurs at moderately low levels of Trp

Translation terminates once the ribosome reaches the stop codon. RNA polymerase has continued transcription into region 4.

Attenuation occurs at moderately low levels of Trp

Short peptide will be degraded

3

Dissociated ribosome

AUG

UGAU

GG

UG

G

1 2

RNA polymerase

Attenuation occurs at moderately low levels of Trp

Once the ribosome dissociates, regions 1 & 2 can and will form a hairpin structure.

Attenuation occurs at moderately low levels of Trp

1-2 hairpin

1 2

34

Attenuation occurs at moderately low levels of Trp

With regions 1& 2 in a hairpin, regions 3 & 4 can form a similar structure

Attenuation occurs at moderately low levels of Trp

UUUUUUUUU

AUG

UGAU

GG

UG

G

RNA polymerase

RNA polymerasedissociates after transcribingpoly U stretch

Attenuation occurs at moderately low levels of Trp

Formation of the 3-4 hairpin, results in transcription termination

Next

Attenuation occurs at moderately low levels of Trp

UUUUUUUUU

AUG

UGAU

GG

UG

G

Attenuation AnimationClick the button to see a faster animated sequence of steps for attenuation just showing the events without all the text.

Attenuation Animation

AUG UGG

RP

Attenuation Animation

1 2AUG UGAUGGUGG

RP

Attenuation Animation

1 2AUG UGAUGGUGG

RP

Attenuation Animation

1 2AUG UGAUGGUGG

RP

3

Attenuation Animation

1 2AUG UGA

3 4UGGUGG

RP

AUG

UG

A

UGGUGG

3

1 2RP

Attenuation Animation

3

AUG

UG

AU

GG

UG

G

1 2

Attenuation Animation

RP

1 2

3 4

UUU

AUG

UGAU

GG

UG

G

RP

Attenuation Animation

Attenuation Animation

1 2

34

UUUUUUUUU

AUG

UGAU

GG

UG

G

RP

Attenuation Animation

RP

UUUUUUUUU

AUG

UGAU

GG

UG

G

AUG UGG

RNA polymerase

Very low levels of Trp prevent attenuation allowing Trp EDCBA genes to be transcribed

The following 5 slides show lack of attenuation in the near absence of TrpClick the button below to view

1 2AUG UGAUGGUGG

RNA polymerase

Ribosome

Very low levels of Trp prevent attenuation allowing Trp EDCBA genes to be transcribed

Ribosome begins translation, but stalls when it reaches 2 trp codons in leader sequence

Very low levels of Trp prevent attenuation allowing Trp EDCBA genes to be transcribed

1 2AUG UGAUGGUGG

RNA polymerase

Ribosome Paused on Trp codons

Very low levels of Trp prevent attenuation allowing Trp EDCBA genes to be transcribed

RNA polymerase continues to transcribe the leader sequence

Very low levels of Trp prevent attenuation allowing Trp EDCBA genes to be transcribed

RNA polymerase

1 2AUG UGA

3 4UGGUGG

Ribosome Paused on Trp codons

Very low levels of Trp prevent attenuation allowing Trp EDCBA genes to be transcribed

Very low levels of Trp prevent attenuation allowing Trp EDCBA genes to be transcribed

1UUUUUUUUU

RNA polymerase2

3

4AUG UGAUGGUGG

Ribosome Paused on Trp codons

Very low levels of Trp prevent attenuation allowing Trp EDCBA genes to be transcribed

Since the ribosome is stalled on region 1, region 2 & 3 are avialable to form a hairpinstructure while RNA polymerase finishes transcribing region 4

Very low levels of Trp prevent attenuation allowing Trp EDCBA genes to be transcribed

1UUUUUUUUU

2

3

4AUG UGAUGGUGG

Ribosome Paused on Trp codons

RNA polymerase

Very low levels of Trp prevent attenuation allowing Trp EDCBA genes to be transcribed

With the formation of the 2-3 hairpin, there is hairpin formed immediately upstream of the poly U stretch.This allows RNA polymerase to continue transcription of the remainder of the operon

Replay

Very low levels of Trp prevent attenuation allowing Trp EDCBA genes to be transcribed