the operon

operon

• group of bacterial genes controlled by a shared promoter - they are transcribed together with their promoter and additional sequences that control transcription

• operator acts as on/off switch - contains specific sequence of DNA which specific regulator proteins can bind to

• produce proteins that bacterium needs at the same time - all genes in an operon are under same control, energy saving mechanism

• inducible operon - transcription is not normally taking place, something must happen to induce transcription

• repressible operon - transcription is normally taking place, something must happen to repress transcription

• 2 types:

catabolic - make enzymes to break things down

biosynthetic - make enzymes to make other substances

polycistronic mRNA

• when multiple genes are all transcribed together to give one long mRNA molecule

• single terminator is present at the group of multiple genes

• genes are then individually translated into their separate proteins

regulator genes and proteins

• regulator gene - not part of operon, helps control expression of structural genes

• regulator gene is transcribed and translated into regulator protein - regulator protein binds to operator and inhibits/initiates transcription

• regulator proteins have 2 binding sites: one which binds to DNA and one which binds to the inducer/repressor

• positive regulatory proteins (activator) - initiate transcription, positive control

• negative regulatory proteins (repressor) - inhibit transcription, negative control

operator

• specific sequences of DNA which specific regulator proteins can bind to

structural genes

genes under control of the operon

negative inducible operon

• controls proteins that break down molecules

• regulator gene codes for an active repressor protein

• repressor protein binds to the operator - operator site overlaps with promoter site

• this means that the repressor protein binding blocks RNA polymerase from binding to the promoter site so transcription cannot be initiated

negative repressible operon

• control proteins that carry out biosynthesis

• regulator gene codes for an inactive repressor protein

• repressor protein cannot bind to operator - RNA polymerase binds to promoter site so transcription takes place

• for transcription to be inhibited, something must happen to activate repressor protein - corepressor binds to repressor

lactose operon

• bacteria can’t use lactose without processing it - enzymes required

• lactose operon encodes enzymes beta-galactosidase, permease and transacetylase

• structural genes: lacZ (beta-galactosidase), lacY (permease), lacA (transacetylase)

• presence of lactose (allolactose) induces operon

• LacI encodes repressor - regulates expression of the 3 operon genes by producing tetramer regulatory protein, binds to operator in absence of lactose to inhibit transcription (blocks action of RNA polymerase)

• Lac promoter structure - operator region overlaps with -10 box, this means that there is steric hindrance

• some of lactose rearranged to allolactose - allolactose (inducer molecule) binds to and inactivates lac repressor so there it does not bind to operator and there is no steric hindrance - RNA polymerase can bind to promoter site and initiate transcription

• leaky process - operon is never entirely off, even with repressor bound there is a low level of transcription, equilibrium

coordinate induction

simultaneous synthesis of several proteins stimulated by an inducer

lactose operon: beta-galactosidase

hydrolyses lactose to produce galactose and glucose which can be used for energy production

lactose operon: permease

transport protein, enables lactose to enter bacterial cell

lactose operon: transacetylase

converts side products of break down of lactose to galactose and glucose inside of cell

lactose operon: mutations of lacY and lacZ

• independent to eachother

• only affect product of gene in which mutation occurs

• partial diploids functioned normally - only one copy of gene required to produce functional enzymes

lactose operon: mutations of lacI

• showed that single copy of lacI gene required to produce functional regulator protein

• lacI+ can be trans acting - restores control even if operon was on a different DNA molecule

• superrepressor mutations - dominant over lacI+, produce repressor with altered inducer binding site so the inducer is unable to bind to the repressor

• lacI- mutation causes constitutive lacZ expression

lactose operator: mutations of lacO

• operator mutations

• alter sequence of DNA at operator so repressor protein cannot bind

• dominant over lacO+ - one copy of lacO+ insufficient for sequence of DNA at operator to not be altered

• showed lacO is cis acting - only affects genes on same DNA molecule

partial diploids

• full bacterial chromosomes with an extra piece of DNA added to the F’ plasmid

• shows the effect of having 2 different versions of a gene on mechanisms within an organism

catabolite repression

• when glucose is available, genes that participate in metabolism of other sugars are turned off - glucose overrides the effect of other sugars

• positive control in response to glucose - catabolite activator protein (CAP)

• cAMP formed from ATP using adenylate cyclase

• CAP needs to bind to cAMP (important in cellular signalling processes) and form a complex before it can bind to DNA

• exponential growth using glucose inhibits adenyl cyclase - less cAMP so less cAMP-CAP complexes formed so less CAP attaches to CAP site - RNA polymerase cannot bind to promoter so lac operon not induced

lactose operon: diauxic growth curve

• additional level of control of lac operon

• diauxic - 2 phases, bacteria grows exponentially then levels off and then grows exponentially again

• glucose - used as initial energy source since it is easier to break down - more energy-efficient, operon control is not used if glucose is present (first stage of exponential growth due to using glucose as energy source)

• beta-galactosidase - activity only begins to rise once all glucose has been used up, second energy source since less energy-efficient, lac operon only induced after nearly all glucose run out (second stage of exponential growth due to using lactose or other sugars as energy source)

the phosphoenol pyruvate: glucose phosphotransferase system

• protein IIC - channel protein, moves glucose into the cell

• protein IIC attached to ‘relay system’

protein IIB

protein IIA

protein HPr

all phosphorylated - involved in regulation of cAMP, phosphorylated protein IIA stimulates adenylate cyclase

• when glucose passes through channel, phosphate donated to glucose from protein IIB

• protein IIA donates its phosphate to protein IIB and protein Hpr donates its phosphate to IIA

• once run out of phosphates, IIA becomes dephosphorylated so that no cAMP is produced as adenylate cyclase is inhibited

how CAP binds to DNA in the promoter

• CAP binding site found in promoter - consists of palindromic sequences

• CAP bends the DNA to force it apart - easier for RNA polymerase to bind since lac promoter -10 box has a G base present

• G base means that strongly binding G-C base pair present so it is more difficult for RNA polymerase to open up a transcription bubble - CAP protein required

extra role of protein IIA

• dephosphorylated protein IIA represses lac permease

• this means that there is less lactose import and less allolactose - more functional lacI repressor so less lac operon transcription

biosynthetic operons

• expression of enzymes that together synthesize small molecules

• if these small molecules are not in the growth medium bacterium has to make them - the operon is switched on

• if these small molecules are in the growth medium bacterium doesn’t need to make them - operon switched off

the tryptophan operon

• biosynthetic repressible operon - transcription normally turned on and must be repressed when the products of its structural genes are not needed

• 5 structural genes - encode enzymes involved in the pathway to make trypotphan

• trpE - contains a long 5’ untranslated region that is transcribed but does not encode a polypeptide - plays a role in another regulatory mechanism

• higher levels of Trp = repressor bound to operator so genes not transcribed, energy efficient since Trp already present so it does not need to be produced

trp repressor

• encoded for by trpR gene - encodes inactive form of repressor (apo-repressor)

• Trp - co-repressor, binds to apo-repressor to form holo-repressor so that it is activated

• Trp apo-repressor has 2 binding sites: one binds to Trp, one binds to operator

• Trp holo-repressor - complete form of repressor, undergoes conformational change to form holo-repressor from apo-repressor when Trp binds - enables repressor to bind to operator so that RNA polymerase is blocked from binding to promoter region due to stearic hindrance