326R
Attenuation
Tryptophan BIO operon review
Operator and promoter overlap
promoter operator
tryptophan trp E trp D trp D trp B trp A
trpR Binding of repressor trpR to operator prevents transcription. trpR By this simple model we would predict that a trpR null mutation (no functional trpR protein) would continuously transcribe this operon at a high rate trpR But thats not what happens
tryptophan
tryptophan
Tryptophan operon: Closer look at Attenuation the transcription unit (Not to scale)
Leader peptide ORF
Leader
rbs
+1
attenuator 1 2 3 4
trp E trp D trp D trp B trp A
RNA from regions 1,2,3 and 4 have complementary sequences Region 2 is complementary to both regions 1 and 3. Region 3 is complementary to Both regions 2 and 4.
Protein coding sequences tryptophan synthesis enzymes 5 ORFs on one mRNA
Attenuator region
RNA from regions 1,2,3 and 4 have complementary sequences Region 2 is complementary to both regions 1 and 3. Region 3 is complementary to Both regions 2 and 4.
1
2
3
4
UUUU
RNA transcribed from this DNA can form two different secondary structures
1
3
1
3
3+4 forms a rhoindependent terminator
2
2
4
UUUU
2+3 structure does not terminate called a preemptor
4
UUUUNNNN
Transcription of the attenuator region
rbs
1
RNA pol
2
3
4
trp E
trp D
trp D trp B
trp A
rbs
rbs
1
1
2
RNA pol
3
4
trp E
trp D
trp D trp B
trp A
rbs
Ribosome binds Shine-Delgarno RBS, begins translation
Translation of the leader peptide during transcription
rbs
1
1
2
RNA pol
3
4
trp E
trp D
trp C trp B
trp A
rbs
Zoom in
Leader polypeptide
ggu ugg ugg cgc acu ucc uga
The region one ORF contains two consecutive trp codons (ugg = trp, uga = stop)
If tryptophan levels are low, then levels of tRNA charged with tryptophan will also be low ribosome will stall when one of the trp codons is in the A site The ribosome is waiting for charged Trp tRNA
ggu ugg ugg cgc acu ucc uga 2 3
X
Incomplete peptide
X
Stalled ribosome prevents base pairing of region 1 to region 2
Region 2 binds to region 3. This prevents binding of 3 to 4 = no termination
High levels of trp allow synthesis of the complete leader peptide Transcription will terminate
ggu ugg ugg cgc acu ucc uga 2
X
3
4
If tryptophan is present, translation proceeds to the stop codon. The ribosome stalls or Releases from there, leaving region 1 uncovered and allowing it to base pair with region 2.
1 binds to 2, prevents formation of preemptor
3 binds to 4, forming rho- independent terminator
Tryptophan operon summary
Regulated by tryptophan levels by two different systems Tryptophan directly binds trp repressor and prevents transcription
Direct: free amino acid is repressor.
Tryptophan - charged tRNAs allows transcriptional terminator to form, stopping transcription
Indirect: sensitive to charged tRNA rather than free amino acid
Regulation by trp repressor protein is more powerful, its able to reduce transcription by about 70 fold, while attenuation only reduces about 8-10 fold.
Together, can they vary transcription by about 600 fold.
Some hypothetical numbers for rates of transcription
Excess Tryptophan: 1 Tryptophan Starved: 700 Excess Tryptophan, null mutation in TrpR: 70 Excess Tryptophan, null mutation in attenuation system: 10
Trp starved vs. non-starved transcription
Tryptophan starved
promoter
1
1
2
2
3
3
4
4
Trp synthesis genes pol
Txn continues to end of Trp synthesis genes
Stalled ribosome (two trp codons)
Preemptor forms, (not a terminator)
Non starved
promoter
1
12
2
3
3 4
4
UUUUU
Trp synthesis genes pol pol
Terminator Ribosome completes tln of leader peptide, stalls at stop codon, allowing 1&2 to base pair
RNA pol released from template due to rho-independent terminator (3+4 hairpin)
Transcription and mRNA from trp-starved cells
Low tryptophan
promoter
1
1
2
2
3
3
4
4
Trp synthesis genes pol
Txn continues to end of Trp synthesis genes
Stalled ribosome (two trp codons)
Preemptor forms, (not a terminator)
Completed, full-length mRNA: polycistronic mRNA with multiple independent translation units (ORFs), each with its own Shine-Delgarno RBS, start and stop codons.
1 2 Leader peptide 3
4
rbs
rbs
rbs
rbs
rbs
Trp E gene
Trp D gene
Trp C gene
Trp B gene
Trp A gene
Beta lactamase regulation by level of ribosomes
(note: Beta lactamase is not needed if the cell is not ATGTAA Terminator S-D/RBS ATG-Beta Terminator growing)
Shine-Delgarno/RBS lactamase ORF-TAA
+1
Transcription
Rho independent terminator
AUGUAA
AUG: start UAA: stop
Start codon for beta lactamase
AUG
mRNA
ORF
stop
High levels of ribosomes lead to increased ribosome occupancy of AUGUAA near terminator Low ribosome levels yield lower occupancy, so the terminator becomes more effective
Beta lactamase
Shine-Delgarno/RBS
ATGTAA Terminator
S-D/RBS ATG-Beta lactamase ORF-TAA
Terminator
+1 Low levels of ribosomes (cell is not growing)
High levels of ribosomes (cell is growing)
txn Ribosome occupies part of terminator sequence
txn
UUUUU
Beta lactamase ORF
UUUUU
Terminator formed, Txn stopped
Terminator not allowed to form, txn proceeds Beta lactamase protein
Bacillus subtilis hutP anti terminator system
terminator +1 5 ORFs for histidine breakdown terminator
Txn Low levels of Histidine
High levels of Histidine
UUUUU Terminator functions, txn stopped before ORFs HutP protein prevents termination ORFs
tln
Five enzymes for Histidine breakdown and utilization
B. subtilis hutP anti terminator system hutP
his his his his his his hutH hutU hutI hutG hutM
hutP protein made at constant low level
hutP
his hutP his his his his his
Polycistronic mRNA encodes genes for histidine breakdown and utilization
Six histidine molecules bind hexameric hutP protein, the complex then acts as a sequence-specific RNA binding protein. hutP- histidine complex binds RNA stem-loop structure and prevents transcriptional termination. Histidine utilization (Hut) genes are then transcribed. If Histidine levels are low, hutP is unable to bind the terminator