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