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Unformatted text preview: Midterm: Thursday evening (12 Feb) Common to all sections Covered material: All including lac operon You can bring: 1 double-sided cheat-sheet Review session: 12 Feb in class. Today we will decide on topics – be prepared and be specific. Postings: Practice Midterm answers Supp. Material on lac (and trp) Quiz questions and answers Regulation of prokaryotic gene expression Example 2: trp operon Tryptophan (Trp) trp operon TrpR repressor Attenuation Leader region Leader peptide mRNA stem-and-loop (hairpins) 2 trp operon
Learning outcomes for the trp operon Learning operon
Students will be able to: 3 Draw a correct representation of the trp operon that includes the genes, the that lleader sequence genes, the promoter and operator regions, and the trpR gene eader gene with its promoter. Draw a correct representation of which proteins are bound to the control regions of the trp operon based on the presence and absence of tryptophan. regions tryptophan Draw and rationalize the alternate structures of the mRNA transcribed from the attenuator region and the impact on transcription of the trpEDCBA genes. the genes. Explain the role of translation of the trp leader peptide on attenuation and leader draw how the ribosome can affect the mRNA structure. Predict the effects of mutations in the DNA sequences of the operon, operon including mutations that affect translation of the leader peptide on the level of trp enzymes in the cell, including full de-repression and basal levels. trp enzymes Use the trp paradigm to explain other examples of transcription regulation. paradigm E. coli metabolism metabolism E. coli contains all the genes necessary to contains 4 synthesize tryptophan from intermediates produced synthesize from during the respiration of glucose. E. coli can also take up tryptophan (Trp) from the external environment. E. coli uses free Trp to create Trp-tRNA, which can Trp-tRNA then be used to add Trp to growing polypeptides then to during translation. How does E. coli regulate tryptophan (Trp) synthesis? How How does E. coli “know” that its Trp levels are low, and it should How levels begin synthesizing proteins to make Trp? begin Trp How does E. coli “know” that its Trp levels are high, and so it should How levels not synthesize proteins that make Trp? not Trp The trp operon and and trpR, the gene for the trp repressor repressor 5 proteins Regulation of the trp operon operon Two levels: Level I: Regulation of the initiation of transcription 6 Similar basis as the lac operon. Involves an allosteric regulatory protein. Binding of the regulatory protein to a small molecule changes its properties. The regulatory protein for the trp operon is the Trp repressor (TrpR), encoded by its own gene (trpR). Comparing lactose and tryptophan
The lac operon and the trp operon strategies differ slightly because: Lactose is a nutrient. The cell gets it from the environment if available when energy and C source are needed. Cell must be able to import it and use it. The operon should only be on when lactose is available as the only C source. Tryptophan (an amino acid) is a product of the cell eventually used by the cell in synthesizing proteins. As such, the cell should only make trp if it is not available from the environment. How will 1st level regulation work for the trp operon?
Regulator Prot 1 Prot 2 Prot 3 Prot 4 Prot 5 U TrpR repressor repressor
TrpR by itself is a dimer, but has no DNA binding activity. dimer When tryptophan is present in the cell, TrpR binds it. binds Bound Trp changes the shape of TrpR to allow DNA binding. to 8 TrpR, the operator, and trp operon repression TrpR the repression
(TrpR dimer + Trp) binds to an operator sequence that overlaps the TrpR Trp promoter where RNAP binds. promoter RNAP can’t bind to the promoter; transcription is inhibited. 9 Note that the TrpR-Trp TrpR-Trp conformation fits nicely into the DNA double-helix TrpR–Trp repression levels TrpR Trp repression
Thus: When there is a high level of tryptophan in the cell, When in TrpR-Trp will reduce the transcription level TrpR-Trp will approximately 70-fold. approximately 70-fold. 10 trp operon – second level of regulation trp
The trp operon has another sequence upstream of trpE, the trpE first gene in the operon. This is called the attenuator region. first operon This attenuator 11 upstream Expanding the attenuator region The leader region of the trp operon mRNA encodes a leader peptide leader and has four segments that can form three possible hairpin structures. and hairpin structures. The leader region of the trp operon operon
mRNA 12 This region contains an open reading frame encoding a 14-amino acid This protein called the leader peptide. protein leader This protein starts with fMet-tRNA followed by two codons for Trp in Trp in the coding sequence. This is a bit unusual (or special), because Trp is a relatively is uncommon AA (only about 1 in 100 AA’s iin a E. coli protein). n protein). To translate the leader peptide, there MUST be Trp-tRNA in the cell. in The leader region of the trp operon operon
mRNA 13 This region of mRNA also has 4 segments that can form hairpin This structures. Three possible hairpins can form, but there are only two options for hairpin combinations. Important note: Some hairpins will act as transcription terminators, others don’t. Leader region mRNA hairpin combinations and outcomes Leader 14 1:2 and 3:4
trpE etc 2:3
trpE etc If the 1:2 and 3:4 structures form: The 3:4 hairpin is a transcription terminator. If this structure forms, RNAP stops just after region 4 before trpE coding region. This prevents transcription of the genes needed to synthesize tryptophan. Leader region mRNA hairpin combinations and outcomes Leader 15 1:2 and 3:4 2:3 If the 2:3 structure forms: Region 3 cannot H-bond with region 4. No transcription terminator is formed. The genes for tryptophan synthesis will be transcribed. How does this work? trp operon – second level of regulation trp
Observation: if the leader sequence of a bacterium is Observation: mutated so that it is missing segment 4, a higher level of tryptophan is synthesized than in normal bacteria. tryptophan is than 16 mRNA What’s going on?? What Level 2: Attenuation in the trp operon operon 17 The additional levels of regulation are provided by stopping The elongation of the trp operon mRNA rather than transcription trp mRNA initiation Transcription is terminated in the “leader region” of the operon. operon This depends on a process involving formation of hairpin transcription terminators. Recall that some hairpins act as transcription terminators and some don’t. Mechanism of attenuation Mechanism
Coupling of transcription and translation! When RNAP transcribes past region 2, the 1:2 hairpin forms. The 1:2 hairpin causes RNAP to “pause” in its elongation, but it does not release the mRNA or DNA. 18 A ribosome can then bind to the mRNA and start translating the leader region. This “nudges” the paused RNAP to continue transcription, and it proceeds to transcribe regions 3 and 4. Mechanism of attenuation Mechanism
Tryptophan levels dictate the outcome! Recall there are 2 Trp codons in the leader peptide. 19 If there is a high level of Trp (thus Trp-tRNA is not limiting), the ribosome will begin to translate the leader peptide and pass through region 1 to region 2. The presence of the ribosome in segment 2 blocks formation of the 2:3 So… termination happens when the level of Trp (Trp-tRNA) is high. This reduces transcription by about 8-10 fold. hairpin, allowing the 3:4 terminator hairpin to form. This leads to termination of transcription. Mechanism of attenuation Mechanism
Tryptophan levels dictate the outcome! If Trp-tRNA is low, the ribosome “stalls” at the Trp codons of the leader peptide and doesn’t block segment 2. 20 This allows formation of the 2:3 hairpin. If region 3 is H-bonded to region 2, this prevents formation of the 3:4 hairpin terminator. RNAP continues past region 4 to transcribe the entire operon. So… the genes for Trp synthesis are transcribed when Trp-tRNA is low Regulation of the trp operon operon
Level 1. Repressor/operator interaction When tryptophan is present, tryptophan binds to the TrpR protein (the product of the trpR gene). TrpR-Trp binds to the trp operator and prevents transcription. Repression reduces transcription of the trp operon ~70-fold. 21 Regulation of the trp operon operon
Level 2. Attenuation: termination of initiated transcripts If Trp levels are high, transcription is terminated before the trpE gene. 22 If Trp levels are low, transcription continues into the trpE gene. Attenuation can regulate transcription levels by a factor of 8 – 10. Combined with the repression mechanism, this allows up to 560-700 fold regulation. PRS questions ...
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This note was uploaded on 11/03/2009 for the course ECON 210 taught by Professor James during the Spring '09 term at UBC.
- Spring '09