Prokaryotic Transcription

Describe the structure of bacterial RNAP

• Exists as core enzyme (alpha, alpha, beta, beta’, omega) and associate with a sigma subunit to form the holo-enzyme

• Core has efficient elongation, sigma for specificity

How does the RNAP holoenzyme stabilize on DNA? Why do we know this?

• Core enzyme has Mg2+ active site at cleft between beta and beta’ subunits that form pincers

• Binds roughly 20Bbp downstream to stabilize binding

• High res x-ray crystal structure

-10 box consensus sequence

• TATAAT

What is the ‘strength’ of a promoter and what does this do?

• How close the sequence matches the consensus seq

• Encodes up/down regulation of a gene - more/less transcripts initiated per unit time

Use of KMnO4 in identifying prokaryotic transcription start sites?

• Unwinds T’s between -10 box and start codon

• Indicates whereabouts in a long stretch of DNA the promoter, and therefore RNAPsigma subunit recruitment, may be

Simply, what are three ways of biochemically mapping of DNA-protein interactions

• Electrophoretic mobility shift assay (EMSA)

• In vitro footprinting

• Chromatin immunoprecipitation (ChIP)

Simply, what does EMSA answer?

• Does a known protein interact with a known DNA fragment?

Describe how to carry out an EMSA, drawing a results diagram

• 32P fluorophore end label known DNA

• Mixed with query protein extract

• Non denaturing electrophoresis to separate by mass

• Control: labelled DNA only column

• If fluorescence of DNA band is greater than at the same point of the DNA/protein mixture column, and a second fluorescent line has appeared, the protein has bound the DNA sequence

https://ibb.co/4kqZCg2

How can an EMSA be carried out to see if specific bases in a sequence are important for protein binding? Draw a results diagram

• Add increasing concentration of unlabelled wt/mutant DNA to labelled wt/mutant DNA bound to the protein of interest (respectively)

• If radioactive signal of labelled DNA:protein remains constant, then the bases mutated are important for protein binding

https://ibb.co/yFJQqbPy

Simply, what does in vitro footprinting answer?

• Where does a known protein bind to on a known long DNA fragment?

Describe how to carry out DNA footprinting, including a results diagram

• 32P label one strand of known dsDNA

• Incubate two samples: one with and one without protein

• Digest mildly using DNase or -OH radicals very mildly so that there are single hits

• Ladder created should be roughly n, n-1, n-2 etc nucleotides due to single hits in random place

• High res non-denaturing PAGE

• Gap is the footprint, where protein binding has shielded from digestion and increased mass so not moved that far

• https://ibb.co/vxmpfJcw

Simply, what does ChIP answer?

• Does this known protein bind to DNA in vivo?

Describe how to carry out ChIP

• Formaldehyde sample under physiological conditions to cross-link any bound protein to its DNA sequence

• Micrococcal nuclease, non-spec cleavage into 200bp fragments

• Protein-A bound antibody to protein of interest

• Immunoprecipitate with magnet

• Reverse cross links

• Sequence

• Locate within genome

Why is ChIP so useful?

• Genome wide

• Very efficient

What is the -35 box consensus sequence?

• TTGACA

How and in what order does RNAP bind the -35 and -10 boxes

• -35 first by sigma4 domain

• -10 after by sigma2 domain

How does the sigma subunit of RNAP cause melting of the promoter?

• Flips A11 and T7 out of their base pairs and into favourable binding pockets on the sigma2 domain

• Does not require ATP

Down mutations at the -35 box inhibit…

• RNAP binding

Down mutations at the -10 box inhibit

• Melting

What is important about the sequences of the -10 and -35 box?

• Asymmetric, ensuring correct polarity of RNAP sigma subunit binding

• Spacing between them is conserved for recognition by sigma2/4 domains (fixed structure) so no ‘scanning’ is required if spacing is maintained

Describe fully how prokaryotic RNAP binds transcription start sites

• Sigma4 binds -35

• Sigma2 binds -10, causes melting and unwinding

• 20bp A/T rich sequence upstream of -35 forms 'up’ element for highly active promoters that can be recognized by the alpha-CTD to stabilize RNAP binding

Describe the transition between transcription initiation to elongation

• First RNA NTP binds with low affinity bping to template strand

• Next NTPs bind with 10x affinity (bp and stacking forces stabilize)

• Cycles of abortive initiation release 2.9nt fragments

• Multiple times before first success

• When reaches beyond 9nt, sigma subunit is lost from complex and transition is successful from this point

What do the multiple rounds of abortive transcriptional initiation reflect?

• Low initial stability of RNA-template strand interactions

• Number of contacts between RNAP sigma and DNA that need to be broken in the correct sequence to keep the RNAP in the correct position, form beta/beta’ clamp required for stable elongation and release sigma

Simply, state four ways transcription can be regulated

• Sigma subunit switching

• Activator/repressor proteins

• Rho independent termination

• Rho dependent termination

Describe sigma subunit switching as a way of regulating transcription

• Sigma subunit of RNAP can be changed in response to the environment

• Stability for alternative sigma increases in their respective environments so they can compete with sigma70 for core RNAP binding

• RNAP recruitment to promoters with different -35 -10 boxes

• Alters what proteins are transcribed

Describe 3 different RNAP sigma subunits

• Sigma70: general transcription

• SigmaN: nitrogen starvation

• Sigma32: Heat shock

• Sigma32 increases transcription of protective proteins that catalyze refolding of misfolded proteins during heat shock responses

Draw a diagram of the full lac operon

https://ibb.co/20Mcjkxp

How do small molecule inducers work to regulate gene expression?

• Activate gene expression by binding inactive activator proteins

• Inactivate by binding active repressor proteins

What is the purpose of an operon?

• Organise regulatory and structural genes together for a coordinated response to a change in environment e.g. what promoters are available

Describe all the components of the lac operon

• LacI promoter - very weak, 2bp match to consensus

• Lac repressor - forms lacI mRNA, encodes repressor proteins that bind second promoter

• CAP site - CAP protein binds to stabilise RNAP alpha-CTDs

• Second promoter - weak -35 and -10, quite leaky for low level transcription of lacZ and lacY

• Repressor binding site - where repressor proteins bind

• LaZ - beta-galactosidase, creates allolactose inducer

• LacY - permease, allows lactose uptake into cell

How are glucose levels measured by the lac operon

• Inversely proportional to [cAMP]

• [cAMP] measured by CAP protein (when cAMP binds, can bind CAP site on operon)

Why is the second promoter on the lac operon being leaky beneficial?

• Basal LacY/Z transcription

• Need some allolactose present so that when glucose does drop and lactose does increase, repressor can be released

• If absolutely no transcription, allolactose scarce so would struggle to release repressor even when needing to switch to lactose metabolism

Draw the diagram showing the binding of activators / repressors to the lac operon in response to different glucose and lactose levels

https://ibb.co/VYgZKSTJ

Simply, in the lac operon what are the small molecule promoters and what do they bind?

• Allolactose, binds active inhibitor (lacI repressor)

• cAMP, binds inactive activators (CAP)

Describe Rho-Independent transcription termination:

• Only factor involved is RNA signal: GC rich hairpin followed by run of 6U

• Forms whilst still inside RNAP exit channel during synthesis

• RNAP stalls

• Helicase activity cannot unfold

• Weak interaction between A and polyU at base of hairpin causes RNA dissociation form template DNA a

Describe Rho-dependent transcription termination

• Rho protein has RNA-dep ATPase that acts as a helicase

• GC rich hairpin followed by run of GCGC forms inside RNAP exit channel

• RNAP paused whilst helicase activity tries to unfold

• Rho translocates along the RNA being formed (using ATP hydrolysis) until reaches RNAP

• RNAP dissociates from template DNA

Key difference between euk and prok RNAP

• Prok RNAP alpha-CTD used for activator binding e.g. CAP

• Euk RNAP II CTD is made of the tandem repeats that allow hypo/hyperphopshorylated states

List 2 key cis elements for prok. transcription and what they do

• -35 box: TTGACA recognised by sigma4 of RNAP

• -10 box TATAAT recognised by sigma2, causes unwinding and melting for initiation

• Determine promoter strength, greater match means higher transcription rate

Draw a diagram showing fully how RNAP binds the mRNA

https://ibb.co/wZFxBkp3 (alpha and beta subunits should have more overlap to show that theyre one holoenzyme - perhaps label this)