1. GEN304 Lecture # 7 Attenuation: Regulation of the Trp operon Reading assignment: pg 374 - 380

2. Key concepts: 1. Regulatory mechanisms for biosynthetic pathway genes. 2. Attenuation is another type of gene regulatory mechanism that is responsive to levels of particular metabolite in cell (Trp) 3. Alternate mRNA secondary structures. 4. Genetic strategies used to elucidate attenuation.

3. lambda uses antitermination to regulate a temporal sequence of gene expression E. coli uses antitermination to prevent premature termination of the rrn operons attenuation is another mechanism for modulating termination that is used for regulating operon expression (Trp, His, Phe...) “ Nature’s objective seems clear: to exploit the various aspects of RNA polymerase function to achieve a multitude of specific regulatory responses” C. Yanofsky

4. Trp operon encodes 5 structural genes, that synthesize tryptophan (essential aa that is required when not supplied in media) Trp operon is regulated by 3 distinct mechanisms, each of which is responsive to levels of tryptophan in the cell BASIC DISTINCTION: catabolic operons (lac, gal, ara): inducible by the substrate of the gene products biosynthetic operons (trp) are usually repressed by the end product of pathway.

5. Trp operon : trp E, D & C: enzymes for indole synthesis (precursor for tryptophan) trp B & A: conversion of indole to tryptophan ** both B and A needed for efficient conversion, but high levels of B alone can do it alone

6. 1. Trp-responsive regulatory mechanisms:

7. note that with catabolic operons, the inducer (eg lactose) acts as a co-activator by “disabling” the repressor lacI in this biosynthetic operon, tryptophan is acting as a co-repressor, without which the repressor is not functional binding of trpR-trp repressor to the operator prevents binding of RNAP to the overlapping promoter - so RNAP can’t initiate transcription

8. 2. tryptophan also acts to inhibit the biosynthesis of more tryptophan by direct feed-back inhibition of the “E” enzyme high trp: E functions poorly, low rate of biosynthesis low trp: E functions efficiently, high rate of biosynthesis trpE trpD indole trpC trpB tryptophan trpA(efficient) trpB indole tryptophan trpA (inefficient)

9. for a long time, these two mechanisms were thought to be the “whole story” of how the Trp operon is regulated - BUT discovery of anomaly (and eventual resolution): work of Charles Yanofsky et al. recovery of mutants that could not be explained by these 2 mechanisms alone …. SO WHAT were the puzzling results that Yanofsky and co found? 2 known facts to start with:

10. if trpE is mutant, cell can’t make tryptophan unless indole is supplied in media. if both trpA and trpE are mutant, then even if cells are supplemented with indole they won’t be able to grow unless trpB is expressed at high levels. Yanofsky used these facts to set up a screen for mutations that would affect trpB, what he was hoping for were mutations that would increase the rate of transcription of trpB

11. Background strain: Screen: mutagenize trpE/trpA mutant cells select for cells that make tryptophan on media that only contains indole as a supplement? (OTHERWISE DIE) up-regulatory mutants of trpB hoped for

12. BIG surprise - mutants mapped between the P/O and the trpE structural gene !! assays done in presence of tryptophan: genotypetrpRtrpB activity WT - 13.3 + (repressed) 0.6 ∂ED2 - 41.0 ** + (repressed) 1.1 ∂ED102 - 99.0 ** + (repressed) 1.3

13. note, “de-repression” in WT means only ~20X increase in trpB levels over repressed levels 3-8 X higher “de-repressed” levels in mutants “repressed” levels are also ~2X higher in mutants (but repression still works) Molecular analysis of these mutants showed them to be deletions, that removed part of the leader region of the trp operon transcript HOW are these deletions increasing transcription of trpB ??

14. Possible explanations: 1. increased transcription rate of trpB - YES. 2. decreased rate of mRNA degradation - YES, but only for ∂102 (not “the” answer). 3. increasing the copy number (eg by gene duplication or DNA amplification) - NO. clue to the nature of this effect: relatively long leader sequence (150 bp) at beginning of operon where mutants mapped in vitro mutagenesis: same result

15. Conclusion: “something” within the leader region influences the rate of transcription of the trp operon genes - WHAT? more detailed molecular analysis of the “leader” sequences [ ] [ [ ] ] ∂ED2 ∂ED102 : in vitro mutagenesis

16. something odd here : within the leader sequence there appears to be a “mini-gene” followed by an intrinsic terminator WHY would there be a rho-independent terminator sequence before the structural genes in the operon? what is the significance of the mini-gene?

17. Structure of intrinsic terminator: the ∂ED2 and ∂ED102 deletions remove this terminator (and the mini-gene) from the leader sequence - is that the key to this problem? what exactly is the mini-gene sequence?

18. mini-gene encodes a 14 aa peptide: Met-Lys-Ala-Ile-Phe-Val-Leu-Lys-Gly-Trp-Trp-Arg-Ser the peptide has 2 tandem Trp codons, so: if a ribosome is going to translate the mRNA for the mini-gene, there have to be tRNAs charged with tryptophan in the cell if tryptophan levels are low, tRNA trp levels are going to be low and ribosomes will tend to “stall” at this position these two alternate states define a regulatory “switch” - Attenuation

19. Sequence of events in Attenuation: 1. Tryptophan levels in cell determine whether or not ribosomes will “stall” within the mini-gene mRNA sequence. 2. Ribosome stalling influences secondary structure of the mRNA (2 alternate configurations are possible). 3. Secondary structure of RNA determines outcome of interaction between RNAP and the intrinsic terminator sequence.

20. Trp leader sequence contains 3 regions that can form hairpin loop structures - 2 alternate configurations are possible.

21. Repressing conditions (tryptophan available): little transcription initiation because of trpR-trp repressor binding to operator not fully repressed though RNAP that does initiate, will recognize terminator and dissociate however “Attenuator”

22. so under repressing conditions, the third element controlling Trp operon expression is negative control by transcript termination as tryptophan becomes limiting, tRNA trp also becomes limiting which means that ribosomes start to ”stall” as they translate the mini-gene. they stall at the site of the trp codons consequence is de-stabilization of the 1/2 loop, allowing formation of the alternate 2/3 loop to form when 2/3 loop forms, the intrinsic terminator sequence is disrupted !

23. Under de-repressing conditions: destabilization of the intrinsic terminator sequence allows RNAP to continue transcribing the Trp structural genes

24. Attenuation control is based on : 1. The amount of charged tRNA trp, which is itself a measure of how much tryptophan is available in the cell. 2. Coupling of transcription and translation. 3. Alternate mRNA secondary structures in the leader region - which will form depends on what is happening to translation of the mini-gene.

25. WHY are both repression (TrpR-mediated) and attenuation used to regulate this operon? not clear, other biosynthetic operons that use attenuation alone for regulation mini-gene sequence His operon:MTRVQFKHHHHHHHPD Phe operon: MKHIPFFFAFFFTFP