Prokaryotic Transcription

Lac operon

First operon discovered is the lac operon

- has three structural genes producing proteins needed to use lactose (sugar)

- cell only wants to make these proteins when lactose around

- if switch from growth on glucose to growth on lactose, lag then growth again

- proteins:

            - galactoside permease – lactose transporter (lacY gene)

- b-galactosidase – breaks b–galactoside bond between Gal and Glu (both together are lactose (lacZ gene)

            - galactoside transacetylase - ? function unknown (lacA gene)

- expression of all three coordinately regulated – induced 1000X in presence of lactose

- the molecule that stimulates transcription of this operon and production of the protein is the inducer (metabolite of lactose for lac operon)

Operon theory

-          a single signal can simultaneously  regulate expression of several genes that are clustered together on a chromosome and are involved in the same process

-          transcribed on one mRNA with multiple ORFs (polycistronic mRNA)

o       for lac operon three ORFs on one mRNA

Experiments that illustrate regulation of the lac operon

1) Mutational analysis and mapping of the structural genes

- Monod and lab made many mutants that can’t grow on lactose

- found a bunch that can’t make enzyme b–galactosidase

            - for these studies, used ONPG and X-Gal as substrates

- easy to assay – can do ONPG in spectrophotometer and X-Gal on plates

- ONPG is cleaved by b–gal to ONP and something else, ONP absorbs at 420nm

- b–gal cleaves X-gal, X-gal is clear but X is blue

            - blue cell colonies

- found a bunch that can’t import lactose and thus lack lactose permease

- all b–gal mutations map to the same place, all lactose permase mutations map to a different place, right next to b–gal mutations

- so they deduced two genes, next to each other on chromosome

2) Evidence for a repressor

- some cells are constitutive mutants – produce b–gal all the time (constitutively)

- many of these mutations map to the same gene (lacI)

- suggests lacI gene encodes a repressor (protein)

- PaMaJo experiment

            - start with mutant cell with no lacI or lacZ genes

- transfer lacI and lacZ genes into the cell on a piece of DNA and grow cells on glucose

            - see b–gal made for a while (1 hr)

            - then it gradually disappears

            - if add lactose, b–gal keeps accumulating

- proposed explanation is that enzyme is produced initially, but after a while enough repressor is produced that it shuts off lacZ txn, unless an inducer is present to prevent lac repressor protein from repressing

- they hypothesized that lac repressor protein binds to a DNA sequence (the operator) and blocks the RNAP

- they also hypothesized that the repressor can change shape when exposed to the inducer to prevent it from binding the operator

Experiments that show that lac repressor protein has two distinct binding domains

-          make mutations in lacI gene

-          two types

o       lacI^-

§         these mutants can never repress the lac operon

§         mutation is in the DNA binding domain of the protein

§         can never bind operator, so can never block RNAP

o       lacI^s

§         superrepressor

§         always represses, even when lactose is present

§         explanation – these proteins cannot bind inducer molecule, so bind the operator all the time, so lac operon can never be transcribed

o       mapping these mutations shows that lacI^- mutations are always at the N-terminal end of protein, lacI^s are everywhere else

§         conclusion is there are two distinct binding domains, one for the operator, one for the inducer

Experiments that show existence of operator sequence

-          some mutations make lac operon constitutive

-          these don’t map to lacI gene, but to the lac operon, right near the promoter

-          lacO^c

Experiments showing cis vs. trans effects

-          both lacI^- and lacO^c mutations make lac operon constitutive

-          how to tell apart?

-          Make merodiploids – partial diploids – by adding a piece of DNA to a cell

-          One exp

o       Start with lacZ+/lacI- cell

o       Add piece of DNA that has lacI+ and lacZ-

o       Cell produces b–gal like wild-type cell

o       This shows that lacI+ is dominant to lacI- (because lac repressor protein can bind operator on a different DNA

-          Second exp

o       Add DNA containing lacI^s gene to WT cell

o       Find lacZ no longer made, even if cell is grown in lactose

o       In this case, mutant is dominant to WT

-          Third exp

o       Cell is lacI+/ lacO^c (so lac operon transcribed all the time)

o       Add lacI+/lacO+/lacZ DNA

o       Cells still constitutive for b–gal expression

o       Because operon with lacO^c is still always transcribed, even though newly introduced operon is regulated normally

-          Cis vs trans

o       If a gene encodes a diffusible element (usually a protein), the dominant allele will override any other allele because it acts in trans

o       If allele is cis acting, it affects only genes on same DNA fragment