Tryp Operon - Copy PDF

TRYPTOPHAN OPERON  The trp operon  lac operon: codes for catabolic enzymes (break down)  trp operon: codes for anabolic enzymes (build up). Negative control of the trp operon : corepressor, aporepressor The trp operon—leader sequence Chorismic a...

TRYPTOPHAN OPERON  The trp operon  lac operon: codes for catabolic enzymes (break down)  trp operon: codes for anabolic enzymes (build up). Negative control of the trp operon : corepressor, aporepressor The trp operon—leader sequence Chorismic acid to tryptophane 11 12 Organization of controlling sites and the structural genes of the E. coli trp operon  Regulation of the trp Operon 1. Two mechanisms regulate expression of the trp operon a. Repressor/operator interaction b. Attenuation (Transcription termination) 2. When tryptophan is present, it will bind to an aporepressor protein (the trpR gene product). a. The active repressor… b. Repression reduces transcription of the trp operon.  Regulation of the trp Operon 3. When tryptophan is limited, transcription is also controlled by attenuation a. Attenuation produces only short (140-bp) transcripts that do not encode structural proteins. b. Termination occurs at the attenuator site within the trpL region. c. The proportion of attenuated transcripts to full- length ones is related to tryptophan levels, with more attenuated transcripts as the tryptophan concentration increases. d. Attenuation can reduce trp operon transcription …  The Trp repressor---the first layer of regulation When tryptophan is present, it binds the Trp repressor and induces a conformational change in that protein, enabling it to bind the trp operator and prevent transcription. When the tryptophan concentration is low, the Trp repressor is free of its corepressor and vacates its operator , allowing the synthesis of trp mRNA to commence from the adjacent promoter. The ligand that controls the activity of the trp repressor acts not as an inducer but as a corepressor. Negative control of the trp operon  Trp operon – Attenuator controlled system CHARLES YANOFSKY  Attenuation---the second layer of regulation The key to understanding attenuation came from examining the suquence of the 5’ end of trp operon mRNA. 161 nucleotides of RNA are made from tryptophan promoter before RNA polymerase encounters the first codon of trpE. Near the end of this leader sequence ,and before trpE , is a transcription terminator, composed of a characteristic hairpin loop in the RNA. The trp operon—leader sequence Chorismic acid to tryptophane 11 12 The hairpin loop is followed by 8 uridine residues. At this so-called attenuator , transcription usually stops, yielding a leader RNA 139 nucleotides long. Three features of the leader sequence: There is a second hairpin (besides the terminator hairpin) that can form between regions 1 and 2 of the leader sequence. region 2 also is complementary to region 3; thus , yet another hairpin consisting of regions 2 and 3 can form and when it does prevent the terminator hairpin (3,4) from forming. The leader RNA contains an open-reading frame encoding a short leader peptide of 14 amino acids, and this open-reading frame is preceded by a strong ribosome binding site. Attenuation in the trp operon The molecular model for attenuation a. Translation of the trpL gene produces a short polypeptide. Near the stop codon are two tryptophan codons b. Within the leader mRNA are four regions that can form secondary structures by complementary base-pairing i. Pairing of sequences 1 and 2 creates a… ii. Pairing of sequences 3 and 4 is a … iii. Pairing of 2 and 3 is an …  The sequence encoding the leader peptide has a striking feature : two tyrptophan codons in a row. The function of these codons is to stop a ribosome attempting to translate the leader peptide. Above all , how transcription termination at the trp operon attenuator is controlled by the availability of tryptophan ? Sequence and structure of the leader  Models for attenuation in the trp operon of E.coli When Trp is abundant: (a) The ribosome continues translating the leader peptide, ending in region 2. This prevents region 2 from pairing with region 3, leaving 3 available to pair with region 4. (b) Pairing of regions 3 and 4 creates a rho-independent terminator known as the attenuator. Transcription ends before the structural genes are reached.  Models for attenuation in the trp operon of E.coli When tryptophan (Trp) is scarce: (a) Trp-tRNAs are unavailable, and the ribosome stalls at the Trp codons in the leader sequence, covering attenuator region 1 (b) When the ribosome is stalled in attenuator region 1, it cannot base pair with region 2. Instead, region 2 pairs with region 3 when it is synthesized (c) If region 3 is paired with region 2, it is unable to pair with region 4 when it is synthesized. Without the region 3-4 terminator, transcription continues through the structural genes The Importance of Attenuation 1. Use of both repression and attenuation allows a fine tuning of the level of the intracellular tryptophan. 2. Attenuation alone can provide robust regulation: other amino acids operons like his and leu have no repressors and rely entirely on attenuation for their regulation. 3. Provides an example of regulation without the use of a regulatory protein, but using RNA structure instead. 4. A typical negative feed-back regulation. Thank you