4 - In Vitro RNAP Reconstitution Flashcards
which RNAP was chosen to be reconstituted and why?
That of M. jannaschii, because it is hyperthermophilic so the protein is more soluble, compact, stable and the N/C terminal regions are folded into the protein making it more biochemically tractable.
What is different about jannaschii RNAPs compared to some other complex ones?
It is in the phylum that does not possess the small subunit Rpo8. The other major archaeal phylum does, as do all eukaryotes.
What are the features of M. jannaschii?
Hydrothermal vent-dwelling hyperthermophile.
Lives at 300atm, 94 degree metal rich environments
autotrophic, only metabolise CO2 to CH2
What are the advantages of reconstituting the protein in vitro?
Purification is simpler, achieves greater purity/yield.
Easy introduction of mutations that would be lethal to the cell.
Can produce RNAPS with molecular probes attached for fluorescence or paramagnetic studies
Can introduce moieties that cause cross linking or cleavage for structural analysis
Culturing jannaschii is dangerous
What approach was taken to reconstituting the protein?
Werner and Weinzierl used PCR to clone the RNAP genes into E coli, each subunit expressed separately. Total denaturation/renaturation approach used.
What are the four largest subunits in the RNAP called?
A’’, A’, B’’, B’
How were the four large subunits expressed and purified, why?
Inclusion body approach as being totally entwined could not fold individually so were insoluble.
Inclusion body solubilised in 6-8M urea and purified with ion exchange chromatography.
Why are urea and guanidine denaturants?
Mimic the main chain backbone of the protein, replacing it and so removing secondary structure. Soluble up to 12M
How are denaturant such as urea and guanidine removed?
Dialysis
What was the main method of purification for the small subunits and why was it used?
heat treatment, as they are soluble individually but are hyperthermophilic proteins so will be the only ones not denatured so easily purified by size exclusion chromatography (S100HR).
Which small subunit was purified differently to the others? How and why?
K, as it is the only insoluble one so could not be heat treated. Instead it was purified by Talon, a kind of His-tag affinity chromatography.
When was the denaturant removed and why?
Only after all the subunit extracts had been mixed together, so that they could fold using each other as templates.
Why must the folded mixture be re-purified, and how is this done?
Many of the complexes will not fold properly, so these must be removed. This can be done using another round of heat treatment/gel filtration, as only the properly folded complexes will not denature.
How was the complete complex identified when it was being purified for the last time?
Transcription assays and silver stained SDS-PAGE analysis of the eluted fractions. Both are necessary as some activity may be shown in complexes other than the complete one.
What criteria must the reconstituted RNAP fulfill in order to be considered valid as a model for study?
All subunits must be present
Active site must be functional (improper folding can inhibit this)
Must interact with TFs properly
Must be able to initiate site-specific transcription
Must respond to transcriptional activators to give a reasonable rate of transcription
How can it be verified that all the subunits are present?
SDS-PAGE
How can it be verified that the complex is active?
Using a non-specific transcription assay
what is a non-specific transcription assay?
The RNAP is incubated with P-32 labeled NTPs and nicked DNA causing it to begin transcribing without a proper start site.
How can it be verified that the complex is capable of initiating site-specific transcription?
By running a transcriptional assay on an SSV T6 transcript, incubating the RNAP and DNA with the archaeal transcription factors TFB and TBP.
What is an example of an archaeal transcriptional activator?
Ptr2
How does Ptr2 work?
two Ptr2 proteins bind to two sites on the promoter and recruit TFB to the promoter. This allows for initiation at promoters where it would otherwise not be possible due to lack of binding stability for TFB
How can it be verified that the complex responds to transcriptional activators?
By using a run-off transcription assay with increasing Ptr2 concentration to show that it does upregulate transcription.
How many stages are there to RNAP assembly?
Three!
What is the first stage of RNAP assembly?
Formation of the Assembly Platform
L D N P
What is the second stage of RNAP assembly?
Formation of the Catalytic Core
B’ B’’ A’ A’’
What is the third stage of RNAP assembly?
Formation of the Auxiliary Subunits
H K E F
What subunits comprise the minimal RNAP?
The assembly platform and catalytic core
L D N P, B’ B’’ A’ A’’
What is the mininal RNAP capable of?
site-specific transcription.
