BIO 504 - Prokaryotic Genome VI - Trp & araC Operons

Trp operon

Prokaryotic repressible operon, aka ON by default, dealing with tryptophan.

• turned off when amount of tryptophan is abundant

• repressor trpR made at a different locus, and binding tryptophan gives it a high enough affinity for its operator site so it can sit down on DNA and stops transcription.

repressible

the Trp opreron in prokaryotes is a ___ system, meaning that it is ON by default, and is only turned off by its repressor protein when tryptophan is abundant.

trpR

trp operon repressor protein

• low affinity for its operator site until bound to tryptophan

• binding tryptophan gives it high enough affinity to sit down on operator site of DNA and block transcription

apo

the ___ form of trpR, aka no ligand bound, makes it so that there is a small space between the 2 helix-turn helix motifs of the protein,

• this space increases when tryptophan binds, thus making enough space for trpR to bind to major groove of DNA

• aka space increases with tryptophan bound so that binding to operator site is possible.

positive regulation

activators allowing transcription to happen/recruiting RNA poly is a form of ____

• example: CAP binding to cAMP and its operator, allowing RNA poly to be recruited to the lac operon

negative regulation

repressor proteins preventing/blocking transcription when bound to operator site

• example: LacI and TrpR

inducible regulation

genes are OFF by default, and need to be induced by something

• example: lac operon

repressive regulation

genes are ON by default, and are repressed by something

• example: trp operon

high lactose, low glucose, CAP-cAMP binding

Under what conditions is the lac operon fully transcriptionally active?

leader sequence/peptide

trp operon includes a ____ downstream of promoter and operator, but upstream of the first protein coding region.

• ___ includes a stop codon right before the first protein sequence of trp operon, leading to the formation of a short sequence with no function that gets degraded.

• One more way to regulate tryptophan formation as it is very expensive to make.

• part of transcriptional attenuation

transcriptional attenuation

Regulatory mechanism controlling prokaryotic transcription via the pairing of specific mRNA sequence to form a transcriptional terminator under specific conditions.

• only works because transcription and translation are coupled in prokaryotes

low

Under __ tryptophan conditions, the first ribosome tethering behind RNA poly will translate 2 successive tryptophan, which will cause the ribosome to stall waiting for tRNA coding for tryptophan molecule.

This wait prevents segment 1 of mRNA to pair with anything, leading segment 2 and 3 to form and anti-terminator hairpin, allowing the mRNA to be translated.

high

Under __ tryptophan conditions, the first ribosome tethering behind RNA poly will translate 2 successive tryptophan, which will does not affect it.

The lack of a pause prevents segment 2 from pairing with anything and thus leads to segment 3 and 4 to form terminator hairpin, blocking RNA poly at the end of the leader sequence

araBAD operon

aka operon that allows bacteria to use arabinose sugar as a source of carbon instead of glucose when glucose level is low and arabinose is present

• has both positive and negative regulation

• inducible system (gene OFF by default)

• regulated by araC protein

araC

protein regulating the araBAD oeron in prokaryotes, made at a locus upstream of the promoter of the operon it regulates.

• repressor sitting on DNA and blocking transcription in the ABSENCE of arabinose

• activator in PRESENCE of arabinose and necessary for RNA poly recruitment

• has 3 operator sites

cis, trans

Binding of arabinose causes conformational changes in araC, favoring __ dimer binding at I1 and I2 instead of _ formation binding I1 and O2 sites by looping DNA.

• binding of ligand = conformational changes = 2 binding terminals can sit next to each other on DNA

CRP

another regulator/activator of the araBAD operon, in addition of araC, which binds to cAMP before binding to its operator and recruits RNA polymerase

presence, absence.

In the ___ of arabinose, AraC binds the promoter in a different conformation and stimulates araBAD transcription (in the ___ of glucose)

stringent response

response used to regulate the ratio of mRNA made relative to the rRNA made

• uses alarmone molecule

• recognize deacetylated tRNA at ribosome site

alarmone

molecule often involved in regulating the stress response in prokaryotes, but also main actor of stringent response

• produced and degraded by RHS proteins

steps of stringent response

1. AA starvation causes deacetylated tRNA build up in cell

2. RelA protein recognize deacetylated tRNA in A site of ribosome and lack of elongation

3. trigger of alarmone synthesis

4. alarmone binds to beta prime subunit of RNA poly

5. triggers destabilization of rRNA promoter complexes thereby inhibiting their transcription/translation

6. results in downregulation of rRNA genes and upregulation of mRNA synthesis