What’s one of the main targets for regulating genes in prokaryotes
Transcription initiation, cuz if its never transcribed we wont even see its product, or if its transcribed a little well see less of the product
Opérons
Opérons are specific to prokaryotes, its basically 1 transcript that contains the coding information for multiple proteins, like multiple genes under the same promoter such that they’re transcribed in bundles
Often metabolic genes that are grouped this way
Are genes on opérons translated one at a time
No, the different proteins can all be translated at the same time since the transcript will have one promoter region but separate ribosome binding sites
Give the basic organization of a prokaryotic gene
Has a promoter region (just upstream of +1), the +1 indicating an open reading frame, the 5’ UTR is non coding so theres usually that little gap, then theres the actual coding sequence starting with ATG aka met
What are some important promoter regions in prokaryotic genes
About 10 nucleotides upstream theres the TATAAT sequence that resembles the TATA box but is NOT the same
There’s also another sequence about 35 nucleotides upstream, TTGACAT
These are important and fairly conserved because they’re RNA polymerase binding sites, first step for transcription
Note: the more conserved the sequence the more efficient/stronger the promoter
Where is the sigma factor from and why is it so important
It’s a subunit in the RNA polymerase and its important because its the subunit that binds the special promoter regions at -35 and -10, TTGACAT and TATAAT respectively, required for proper positioning of the machinery
Why can RNA polymerase bind so easily to DNA in prokaryotes as opposed to in eukaryotes
In eukaryotes DNA exists wrapped around histones forming nucleosomes, semi bundled up, never straight
In prokaryotes, genomic DNA is more or less naked, not so much interference, so as long as you have a solid -35/-10 promoter sequence you’ll have fairly easy binding, thus why the default setting of transcription tends to be on, because its so accessible
How specifically is transcription regulated in prokaryotes
The ability of RNA polymerase to bind is modified using other proteins, so if a protein wants to “help” RNA polymerase bind or stay attached to the promoter sequence, we call it an activator
If the protein is trying to block a bound promoter by standing in its way, its a repressor, wont allow transcription to occur
What do repressors of RNA polymerase bind
They bind a specific region called operator, literally a specialized binding site for repressors
What is an inducer
An inducer is an effector that increases gene expression, can do so either by positive or negative mechanisms
The effector can either activate an activator to increase gene expression or it can inhibit a repressor, both work
These are forms of allosteric regulation
What is allosteric regulation (conceptually)
It’s a binding of a molecule, an effector to an allosteric site, which is not the main binding site, and the binding of this effector will have a conformational change on the protein such that it will behave differently, either more or less effective
In the case of RNA polymerases, it’ll change the proteins binding affinity to DNA
This induced change is an allosteric transition
What are the components of the Lac opéron
Has 1 regulatory component, has a binding site for repressors as always, and it has 3 structural genes, Z, Y and A, in that order, we dont really care for A but we do care about the proteins encoded in Z and Y, Beta gal and permease respectively, and theres also I (like i) upstream, encodes for the repressor
This operon is basically making the uptake of lactose in extracellular environment possible
Beta galactosidase
Protein encoded by Z gene in Lac operon, basically cuts lactose molecule into 2 parts, the galactose half and the glucose half
How is the Lac operon repressor regulated
In the absence of lactose, the repressor is left unchecked and will go inhibit the transcription of this operon (makes sense why would you make protein to digest something not in your environemnt)
But in the presence of lactose, the effector, the repressor will be inhibited and transcription will occur, so lactose is an inducer in this case
What are the classes of Lac mutants
Inducible mutants: where either Beta Gal or permease doesnt work but the other is inducible with IPTG
Non-inducible: where it cannot make either, not even in the presence of IPTG (regulatory mutation)
Constitutive: levels of both were high even without lactose present, present all the time
Partial diploids
It was basically the insertion of a plasmid containing a second copy of a gene, in this case a lac gene, such that you can test dominance vs recessiveness
Partial because is not the whole genome thats diploid and note this was in bacteria so traditional crossing wasn’t possible
This test allowed them to determine whether they were cis or trans acting factors
Cis vs trans acting factors
Cis as in only affecting transcription of genes on the same DNA molecule
Trans as in also being able to affect the transcription of genes on the other DNA molecule
2 in this case due to the second plasmid
What makes the super repressor of the Lac operon so effective
The fact that in has a mutation in the binding site for the effector, so even if the cell wanted to inhibit it it couldnt bind the effector
Also explains why having 2 copies didnt rescue
Are the operator, promoter and repressor regions cis or trans for the lac operon
Operator and promoter are cis, only affecting their own DNA an ether repressor gene, the i region, is trans so affects everyone
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