Lac operon- problem solving PDF

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Lac operon- problem solving...

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The lac operon: an example of a transcriptionally regulated system The most direct way to control the expression of a gene is to regulate its rate of transcription; that is, the rate at which RNA polymerase transcribes the gene into molecules of messenger RNA (mRNA). E. coli break lactose down using two (there are 3, but our problem set only addressed two) enzymes: beta-galactosidase, which is encoded by the lacZ gene, and permease, which is encoded by the lacY gene. These genes and the regions that regulate them are called the lac operon. Other important players in the lac operon are: Operator (lacO) the binding site for the repressor Promoter (lacP) the binding site for RNA polymerase Repressor (lacI) the gene encoding for the lac repressor protein- in the absence of lactose the repressor protein binds to the operator and blocks binding of RNA polymerase at promoter The genotypes are written in order of: repressor (i), promoter (p), operator (o), lacZ (z), lacY (y). Wildtype (‘good copies’) of genes are indicated by a + sign. Example: lacI+ lacP- lacO+ lacZ+ lacY+ If the organism in question is a partial diploid, the two chromosomes are written side by side, separated by a slash: lacI+ lacP- lacO+ lacZ+ lacY+/ lacI- lacP+ lacO+ lacZ- lacY+ How to solve a lac operon question. Look at the promoter first- do you have a good copy of the promoter (lacP+)? If not, RNA polymerase cannot get transcription started and that operon is a bust. If you do have lacP+, next look at how the repressor and the operator interact. Here are a few scenarios: lacI+ lacP+ lacO+ the repressor binds to the operator in the absence of lactose and inhibits transcription of the z and y genes. BUT, when lactose is around, it kicks the repressor off of the operator, and the z and y genes are expressed (look to see whether you have good copies of z and y, i.e. z+ and y+). lacIs lacP+ lacO+ this is a super repressor- lactose cannot kick it off, so transcription of z and y FAILS whether lactose is around or not. lacIs lacP+ lacOc the super repressor meets the constitutive operator. The constitutive operator deflects any repressor (even a super repressor) and transcription OCCURS whether lactose is around or not. One last thing to remember is that while lacP and lacO are regulatory regions and only affect the genes that they are sitting right next to (they act in cis), LacI codes for a diffusible regulatory protein, so it can affect both chromosomes (it can act in trans) of a partial diploid. An example lacIs lacP- lacOc lacZ- lacY+/ lacI- lacP+ lacO+ lacZ- lacy- in this case, the first chromosome is a bust because of a bad promoter. The second chromosome has a bad repressor, but the protein produced by the super repressor from the first chromosome will bind to the operator of the second chromosome. Transcription is thus blocked in both operons in the merodiploid.

lac operon Element ß-gal (lacZ)

lacZ+ lacZPermease (lacY) Structural genes

lacY+ lacYTrancetylase

(lacA)

Affects the structure of enzyme rendering it nonfunctional Gene that encodes for permease that allows lactose to enter the cell Normal gene Affects the structure of enzyme rendering it nonfunctional Unknown function

lacA+

Normal gene

lacA-

Affects the structure of enzyme rendering it nonfunctional Binding site for repressor

Operator (lacO)

lacO+

Normal operator

lacOc

Constitutive operator, repressor cannot bind (even a super-repressor) Binding site for RNA polymerase

Promoter (lacP)

Regulatory genes

Purpose Gene that encodes for enzyme that cleaves lactose Normal gene

lacP+

Normal promoter

lacP-

RNA polymerase cannot bind

Repressor (lacI)

lacI+ lacI

-

lacIs

cis

Gene encoding lac repressor protein Normal repressor trans Repressor is not produced Super-repressor, repressor cannot be inactivated by inducer

Cis-acting: Affects only genes on the same DNA molecule. Trans-acting: Codes for a diffusible product, affects genes located on a different DNA molecule....