Prokaryotic Transcription Regulation Flashcards
How do you alter RNAP
substitute sigma factors - classes of genes can be co-ordinately controlled by this.
sigma factors - 7 s subunits in E. coli
Heat Shock: short term switch off of general gene expression. Translation efficiency and protein stability s32 increased. s32 recruited RNAP to promoters w diff -35 and -10 boxes. switches on expression of 20 protective proteins - catalyse correct refolding of proteins
what is induction/repression
substrate or small molecule is an inducer. if available (lactose), metabolising enzymes switched ON (induction)
small molecule is a repressor. if nutrient available (Trp, Arg) , endogenous biosynthetic enzymes switched OFF.
inducers and co-repressors are allosteric regulators of activator and repressor proteins.
reg proteins two conforms: stabilised by inducer or co-pressor, other binds DNA w high affinity. often bind as dimers
What is the Lac operon
reg and structural genes organised into operon to allow co-ordinated response.
regulated promoter LacZ, Y and A fully switched on when Lactose available AND glucose[low] so [cAMP] elevated.
Lac operon has 2 promoters - weak constitutively active promoter (-35 box weak) that transcribes LacI mRNA - encodes Lac repressor. regulated lac promoter as above. glucose easier substrate.
low basal levels of transcription mean that enough LacY product. side reaction of B-galac = inducer that binds to lac repressor
max levels of transcription require activator - CAP protein, -10 and -35 elements reg promoter sub optimal
binding site for CAP protein is upstream of -35 box.
how is lac operon is negatively/positively regulated
by lac repressor negatively, catabolite activator protein (CAP) positively.
if (-lac, +/- cAMP) - repressor blocks RNAP binding
(-cAMP, + lac) - inducer binding releases repressor from operator RNAP binding weak
+cAMP, +lac - inducer binding releases repressor from operator. CAP+cAMP binds DNA and promotes RNAP binding.
whats some evidence
effects of mutations upon lac promoter activity in vivo assay by B-gal activity.
how Negative regulation?
Lac repressor: tetramer of 38kDa subunits - dimer of dimers
Operator recog by N-terminal helix-turn-helix motifs of one dimer docking into major groove
additional repressor binding sites. one required for full repression
repressor binding to operator blocks RNAP binding. inducers such as allolactose bind repressor and reduce affinity for operator DNA
Natural inducers metabolised by B-Gal, so activation only persists while lac available
repressor only active at Lac operon
what is importance of spec and non-spec binding
affinity for non-spec DNA is sufficient for 99.99% repressor to be DNA bound.
non-spec binding accelerates binding to spec sequences by 100-1000 fold. expected rate lower than measured rate.
non-spec binding restricts effective volume in which protein diffuses - smaller volume to search for operator = Facilitated transfer
facilitated transfer = sliding along DNA and/or intersegment transfer
