Genet 270 - topic 2 trp operon

is the trp operon catabolic or biosynthetic?

Biosynthetic (anabolic) - encodes enzymes for synthesizing L-tryptophan

what regulatory role does tryptophan play in the transcription initiation of its own operon?

Co-repressor → binds to the TrpR aporepressor → changes its conformation so that it GAINS DNA binding ability and represses the operon

what is attenuation and where does it occur in the trp operon?

mechanism of transcription termination

occurs prematurely in the leader region (before structural genes)

what is the key feature of the leader peptide that allows the cell to sense tryptophan concentration?

leader peptide contains two adjacent Trp residues

under high tryptophan levels, how does the mRNA secondary structure cause attenuation?

ribosome rapidly translates the leader peptide, allowing segments 3 & 4 to pair → forms intrinsic terminator hairpin

under low tryptophan levels, how is attenuation prevented?

ribosome pauses at the adjacent Trp codons, allowing segments 2 & 3 to pair → blocks formation of the 3:4 terminator

what are the controls of biosynthetic operons (Trp)

aporepressors and corepressors

what is the main difference between trpR and LacI

TrpR protein only binds operator if aa tryptophan is present

what is the function of trpR

encodes repressor protein then binds to operator and occludes RNAP → TrpR changes its conformation so it can bind DNA and repress operon expression

how are catabolic and anabolic operons BOTH regulated

both regulated by repression

what is the function of a catabolic (degradative) operon

to degrade compound X

what is the function of an anabolic (biosynthetic) operon

to SYNTHESIZE compound X

how is expression controlled in catabolic operons

expression is INDUCED by compound X

how is expression controlled in anabolic operons

expression is REPRESSED by compound X

what type of regulatory protein controls both catabolic and anabolic operons

a repressor

what effector molecule is used in catabolic operons

an inducer

what effector molecule is used in anabolic operons

a corepressor

what happens to the repressor in catabolic operons when the effector (inducer) is present?

the repressor LOSES DNA binding ability

what happens to the repressor in anabolic operons when the effector (corepressor) is present?

the repressor GAINS DNA binding ability

which proteins exemplify these systems (catabolic and anabolic)

LacI = catabolic, lactose operon

TrpR = anabolic, tryptophan operon

what are the two states of repressors in anabolic pathways

1. aporepressor = repressor in absence of effector/co-repressor

2. co-repressor = effector molecule binds aporepressor, enabling DNA binding

what does the trp operon encode

enzymes for synthesizing L-tryptophan (an amino acid)

how many genes are transcribed from the trp operon promotor (Ptrp)

5 genes from a single promoter

where is the TrpR repressor encoded relative to the trp operon

at a separate, unlinked location in the genome

how is trp operon expression regulated by tryptophan concentration

1. high tryptophan = operon NOT expressed (repression)

2. low tryptophan = transcription of trp operon turned ON

how does the regulation of the trp operon differ from the lac operon

trp operon: tryptophan acts as a CO-REPRESSOR = repression

lac operon: lactose acts as an INDUCER = induction

what is the effector molecule for the trp operon

tryptophan (co-repressor)

what is the effector molecule for the lac operon

lactose (inducer)

does trp operon regulation involve cAMP-CRP

no regulation by cAMP-CRP

what are the 2 steps of trp operon expression

1. control of the trp operon expression at transcription initiation

2. control of the trp operon expression at transcription termination

what is attenuation in the trp operon

regulatory mechanism where transcription starts but is terminated before RNAP reaches the first structural gene

why does attenuation occur?

because ribosome movement during translation affects the secondary structure of the mRNA leader region

what type of control does attenuation provide?

graded control of gene expression levels (in proportion to intracellular tryptophan concentration)

what genetic evidence supports attenuation in the trp operon?

• trpR mutants show higher enzyme levels even without tryptophan → suggests second mechanism

• mutations lowering tRNA Trp increase operon expression

  • deletion of the trp leader region abolishes attenuationwhat ha

what happens in the presence of excess charged tRNA Trp

transcription stops in the leader region due to attenuationwhat

what happens when tryptophan levels are high (but not enough for TrpR repression)?

ribosome translates smoothly → 3:4 hairpin forms→ transcription terminates (attenuation)

what happens when tryptophan levens are low?

ribosome stalls of Trp codons → 2:3 hairpin forms → prevents 3:4 terminator → transcription continues

what structural features are in the trp leader region?

• four sequence regions (1-4) that can form alternative hairpins (1:2 & 3:4 vs. 2:3)

  • a leader peptide with two adjacent Trp codons

how does attenuation differ from repression by TrpR?

• TrpR repression = binary ON/OFF control (tryptophan as co-repressor)

• attenuation = fine-tuned, graded control based on translation dynamics

how does the trp leader region cause attenuation?

by forming an intrinsic transcription termination structure (HAIRPIN) when tryptophan levels are HIGH

what determines whether the terminator forms in the trp leader region?

the rate of the leader peptide translation → depends on intracellular tryptophan concentration and tRNA trp availabilitywhat f

what factors influence attenuation in the trp operon?

• concentration of tryptophan (alters charged tRNA trp levels

• rate of leader peptide translation

• alternative mRNA secondary structures in the leader region

  • effects on RNAP progressionwhat ha

what happens after RNAP transcribes regions 1 & 2 of the leader?

a stem-loop forms → RNAP pauses → ribosome begins translating the leader peptidewhat

what occurs when tryptophan is sufficient (tRNA Trp plentiful)?

• ribosome translates leader peptide fully to stop codon

• ribosome blocks region 2

• regions 3 &4 pair → terminator hairpin forms

• transcription terminates early (~140 bp transcript) → attenuation

what occurs when tryptophan is insufficient (tRNA Trp scarce)?

• ribosome stalls at Trp codons in leader peptide

• region 2 remains free → pairs with region 3

• 2:3 hairpin forms (anti terminator)

• prevents 3:4 terminator formation

  • RNAP continues→ remainder of trp operon transcribed

what is the functional difference between the 2:3 and 3:4 hairpins?

2:3 hairpin → ant-terminator → transcription continues

3:4 hairpin → terminator → transcription stops

why is attenuation considered a fine-tuning mechanism?

because it provides graded control of gene expression based on translation dynamics, not just on/off expression

what happens if mutations prevent formation to the 2:3 hairpin in the trp leader region?

ribosome cannot initiate translation → hairpin 1:2 persists → 3:4 hairpin forms → constitutive termination

whats the difference between regular termination and constitutive termination?

regular termination = conditional, regulated by tryptophan levels and ribosome behavior

constitutive termination = unconditional, caused by mutations, termination occurs every time, independent of tryptophan concentration (always terminates)

what is the effect of mutating the AUG start codon of the trp leader peptide?

ribosome cannot initiate translation → hairpin 1:2 persists → 3:4 hairpin forms → constitutive termination

why does the AUG start codon mutation cause constitutive termination?

without ribosome stalling at Trp codons, region 2 is unavailable → 2:3 hairpin cannot form → 3:4 terminator always forms

how does amino acid starvation (eg. arginine) in other operons provide evidence for attenuation?

ribosomes stalls at codons in the leader peptide → similar attenuation mechanism observed in the arg operonwha

what does this genetic evidence collectively demonstrate about attenuation?

attenuation depends on ribosome position and leader peptide translation dynamics not just TrpR repression