1. Regulation of Gene Expression in Bacteria (Trp operon) Fahareen-Binta-Mosharraf MNS

2. The trp Operon

3. Trp Operon  Trp-tryptophan  The genes of the trp operon are used to make the aminoacid tryptophan.  It is turned off when enough tryptophan is in the cell. Tryptophan is the effector molecule  Operon codes for 5genes–found in E.coli  Five polypeptides combine to make three enzymes.  Each enzyme participates in a step to make tryptophan

4. The trp Operon of E. coli 1.If amino acids are available in the medium, E. coli will import them rather than make them, and the genes for amino acid biosynthesis are repressed. When amino acids are absent, the genes are expressed and biosynthesis occurs. 2.Unlike the inducible lac operon, the trp operon is repressible. Generally, anabolic pathways are repressed when the end product is available.

5. Gene Organization of the Tryptophan Biosynthesis Genes Trp operon spans ~7 kb and produces 5 gene products required for synthesis of the amino acid tryptophan. Trp operon contains 5 biosynthetic coding genes, trpA-E. Promoter and operator are upstream of trpE. Leader region (trpL) occurs between trpA-E coding genes and the operator. Within trpL is an attenuator region (att). TrpR (repressor protein gene) occurs upstream of the promoter

6. Fig. 16.15 Organization of controlling sites and the structural genes of the E. coli trp Trp operon architecture Overlapping genes

7. Concept of Attenuation - When an RNA molecule can take up alternative secondary structure by utilizing different schemes for intramolecular base pairing. This type of mechanism can be used to regulate the termination of transcription - Attenuation: Describes the regulation of bacterial operons by controlling termination of transcription at a site located before the first structural gene. - Control of gene expression by premature termination of transcription - Attenuator: Is a terminator sequence at which attenuation occurs

8. The principle of attenuation is that some external event controls the formation of the hairpin needed for intrinsic termination. If the hairpin is allowed to form, attenuation prevents RNA polymerase from transcribing the structural genes. If the hairpin is prevented from forming, RNA polymerase elongates through the terminator and the genes are expressed Repressor lowers transcription 70-fold (as compared to derepressed state)  attentuation permits another 10-fold control  total dynamic range of control = 700-fold

9. Regulation of the trp operon:  Two mechanisms regulate the trp operon: 1. Repressor/operator interaction 2. Termination of initiated transcripts 1. Repressor/operator interaction When tryptophan is present, tryptophan (corepressor )binds to trpR gene product (inactive repressor-aporepressor). The active repressor (aporepressor plus tryptophan) binds the trp operator, and prevents transcription initiation. trpR protein binds to the trp operator and prevents transcription. Repression reduces transcription of the trp operon ~70-fold.

11. Tryptophan Operon - Repressor Binds when tryptophan is present

12. 2. Termination of initiated transcripts Transcription also is controlled by attenuation, process of translating a short, incomplete polypeptide. att sequence contains a start codon, 2 Trp codons, a stop codon, and four regions of sequence that can form three alternative secondary structures. When cells are starved for tryptophan, trp genes are expressed maximally. Attenuation can regulate transcription level by a factor of 8 to 10, and combined with the repression mechanism, 560-700 fold.

15. The molecular model for attenuation a. Translation of the trpL gene produces a short polypeptide. Near the stop codon are two tryptophan codons. b. Tight coupling of transcription and translation in prokaryotes makes control by attenuation possible.

16. The molecular model for attenuation

24. High Tryptophan- tRNA -->> Terminates Transcription Low Tryptophan- tRNA -->> Transcription Continues Translation Event controls Transcription Event

25.  RNA polymerase pauses when regions 1 and 2 base pair just after they are synthesized  During the pause, a ribosome loads onto the mRNA and begins translation of the leader peptide