Gene Regulation
I. Transcriptional Regulation
Operons (clustering by function)
regulatory region of an operon
promoter
operator
leader region
Negative regulation
repressor proteins (may bind corepressor molecules)
Positive regulation
activator protein (may bind coactivator molecules)
II. The lac Operon (Negative Regulation)
Jacob and Monod proposed the operon model
lacI, operator, promoter, lacZYA
LacI, when bound to allolactose, binds operator to inhibit transcription initiation
Isolation of Common Mutants
lac- mutants could not grow on lactose
lacc mutants lac genes were always expressed, whether lactose was present or not
Defining the Operon (complementation tests)
common recessive lac- mutations that complements other lac- mutations...
...gene products act in trans
defines structural genes (such as lacZ-, lacY-)
rare recessive lac- mutations that don't complement any complementation group...
...probably act in cis and could be a...
-mutation in the promoter/operator region
-polar mutation in first gene of the operon
rare dominant lac- mutations...
...all map to lacI
called lacIs (superrepressor)
common recessive lacc mutations...
...all mapped to lacI
inactivated lacIrare dominant lacc mutations...
...all mapped to lacI
inactived LacI protein but it could still form tetramers
As a Tool in Molecular Biology
lac promoter is inducible. Allowing production of toxic genes
IPTG, nonclevable derivative of allolactose
Several colorimetric substrates exist for the lacZ gene product, beta-galactosidase
ONPG turns yellow in the presence of beta-galactosidase
X-gal turns blue in the presence of beta-galactosidase
III. The L-ara Operon (Positive Regulation)
Operon Structure
araC, operators, inducers, araBAD
Isolation of Mutants
ara- mutants are still common but..
arac muants are rare
because the mutation must make AraC active without binding arabinose
Inactivation of araC (unlike lacI) produces an ara- phenotype
AraC must also be an antiactivator since...
araCc mutations should be dominant (but they are not).
IV. The trp operon (Negative regulation and transcriptional attenuation)
Negative regulation by trpR gene product
TrpR only binds/represses operon when binding tryptophan...
...so TrpR is an aporepressor
Transcriptional attenuation
Leader sequence encodes a
14 amino acid leader polypeptide not a 'functional' protein, but acts to regulate transcription
2 trp codons at position 10 11 are key
Four segments in the leader RNA that can form secondary structures
Bottom Line of regulation
if segments 3 and 4 form a hairpin. a transcriptional terminator is formed...
...and transcription terminates
if segments 2 and 3 form a hairpin, the transcriptional terminator cannot
...form and transcription will continue
The result is that tryptophan levels are regulated as a gradient rather than just on or off