Lecture 17 Flashcards
Clones of three adjacent genes involved in arginine biosynthesis have been isolated from a bacterium.
You have probes for each gene.
Design an experiment to test whether they are part of an operon:
from 3 possible experimental approaches which should you chose
(a) southern blot
(b) northern blot
(c) western blot
B → northern blot → RNA → single mRNA transcript → if not operon → 3 bands
Southern blot → DNA
Western blot → Proteins
Inducible operon
normally off
must be turned on/ induced
ex. lac
Repressible operon
normally on
must be turned off
ex. Trp
Repressor
regulatory protein
bind to operator to turn off transcription
Operator
DNA cis acting element
bound by repressor
Operon
set of regulatory sites and adjacent structural genes
One promoter, one transcript, multiple proteins
E. coli trp operon is
repressible
Repressible systems keyed to end products
Tend to have ____ functions
Transcription stopped/slowed when ____
Stopped by ___
(ex. Paradigm is trp operon in E. coli)
biosynthetic functions
expression not needed
repressor → often the end-product of the biosynthetic pathway → controlled by operon
Transcription of trp structural genes is keyed to the end product ___
tryptophan
aka trp operon enzymes → produce tryptophan
trp operon controls tryptophan biosynthesis → steps DNA to RNA to Protein
1) polermerase → NOT transcribed
2 ) operator → NOT transcribed
3) trpL → leader seq → IS transcribed
4) structural genes → enzymes → required for biosynthesis of tryptophan
Two levels of repression in trp
(i) Feedback inhibition
(ii) Attenuation
Feedback inhibition (in trp)
inhibition is where → high levels of end-product shut down biosynthesis
end-product is tryptophan
→ is a co-repressor that → binds and activates the repressor
tryp absent → repressor not bout (but still around)
tryp present → bind to repressor → conf. change → repressor bind to trpO
Bacteria sence presences of tryptophan and shut down expression of enzymes in order to
not waste energy
mRNA synthesis in prokaryotes vs eukaryotes
Prokaryote → no nucleus → ribosome immediately binds (simultaneously both → time and space)
Eukaryote → mRNA processed and exported to nucleus
transcription termination sites
are GC-rich inverted repeats followed by 6+ A’s on template strand → makes hairpin loop → how know when done transcribing /signals stop transcription
transcription vs translation
transcription → DNA to mRNA
translation → mRNA to protein
Attenuation (in trp)
Fine-tunes transcription after initiation
translation of mRNA leader is a system for sensing tryptophan levels and
controlling transcription of trp structural genes (E, D, C, B, A)
High [tryptophan] halts transcription downstream
Low [tryptophan] allows transcription to continue
in absence of the trp repressor protein,trp mRNA leader is
transcribed and translated
Trp mRNA leader has 2 key features
(i) encodes a peptide that senses [trp]
(ii) forms alternative 2(secondary) structures
4 regions in leader of trip operon
1 → short peptide → two tryp residues
2 → seq complemetarty to seq (in region 3) → bp form secondary structure
3 → same as two → forms secondary structure with 4
When tryptophan is abundant
1 → RNA pol
2 → binding of ribosome
3 → region (1) has two tryp residues
4 →ribosome sits on top of regions (1) and (2) (no pb binding)
5 → when this happens (3) and (4) make hairpin → initiate termination (RNA pol disassociates)
When tryptophan is low
1 → ribosome stalls (on region 1)
2 → region (2) is available to bp with region (3)
3 → anti termination complex → facilitates continued transcription
Pace of translation determines ____ which _____
leader structure which → controls transcription downstream
low tryptophan levels, pace of translation
is slow
high tryptophan levels, pace of translation
is fast
- Will downstream transcription of trp operon genes occur?
→ No. The ribosome will not stall at the 2 gly codons. This ”mimics” tryptophan abundance. Transcription terminates. - What if the 2 trp codons were changed to stop codons?
→ The ribosome stalls at the stop codons (it cannot continue past them). This mimics tryptophan starvation. Transcription continues.
Why is attenuation unique to prokaryotes
i.e., not found in eukaryotes?
attenuation depends upon the fact that transcription is coupled to translation
