Prokaryotic txn regulation II

Observation:

E. coli grown in glucose have nearly no txn of lac operon.  Cells grown in lactose have tons of expression.  Cells grown in glucose+lactose have very little expression – more than in glucose alone, but 100X lower than in cells grown on lactose.  Why don’t cells grown on glu+lac have as much lac operon txn as cells grown on lactose?

Upstream of lac promoter is CRP (cAMP receptor protein, also known as CAP) binding site

-          CRP is a protein

o       Binds the lac operon upstream of -35 box

o       DNA binding activity of CRP is regulated

§         When cells are grown in glucose, cAMP levels in cell low, cell is happy

§         E. coli grow better in glucose than on any other sugar, so even if other sugars are present they want to use glucose

§         When cells are grown in medium lacking glucose, cAMP levels in cell rise, cells are unhappy (not much ATP) and high cAMP is a danger signal

§         cAMP binds CRP protein, now CRP-cAMP can bind DNA

§         binds to bunch of catabolic operons so a bunch of operons are induced

·         these operons are for the utilization of other sugars (maltose, arabinose, cellobiose, etc)

·         all these operons encode proteins that are used to use sugars as an energy source – just like lac operon!

-          CRP actually contacts RNAP

-          Activates transcription

o       By increasing binding of RNAP to promoter

o       Lac promoter has -10 and -35 boxes not that close to consensus

o       CRP and RNAP can bind cooperatively (more protein/DNA contacts = more binding)

o       So more transcripts produced

Summary of lac operon regulation

Using lac operon parts in biotechnology and research

Using lac Z as a reporter gene

-          reporter gene encodes a protein whose level in the cell is easily, sensitively, and cheaply quantifiable

-          can measure amount of txn of any gene by Northern blot or microarray

o       Northerns are time consuming and difficulty

o       Microarrays are expensive and take time

-          But if I fuse lacZ gene to txn control region of any gene, (usually on a plasmid), that txn control region will produce lacZ mRNA and b-gal

o       Easy to assay amount

-          When would you use?

o       Studying txn control regions of a gene – easy to stick region on plasmid, fuse lacZ to it, meaure b-gal activity

o       ID a set of genes regulated by the same stimulus

§         As in book with Ecoli genome and UV as stimulus

o       Look for tissue specific expression in eukaryotes

Using lac operon upstream region to control gene expression of some protein

-          regulated and highly expressed when induced

-          if want to make human growth hormone in E coli, fuse cDNA to lac control region

o       grow up cells in glucose

o       induce txn using IPTG (artificial inducer)

o       gets tons of hGH, all at once, so if bad for the cell, who cares?

Lac operon questions:  3, 5-8, 10-12, 14ab, 16, 17  19 seems good too, but it was too long for me to really go through.  Lots of these are the same idea, but they all help you understand the lac operon.