L10: Gene regulation and transcription factors in bacteria

where do RNA polymerases and transcription exist in bacteria

cytoplasm

polycistronic genes

• bacterial genes are organized into these

• produce a single mRNA molecule from a single promoter

mRNA from polycistronic genes

a single gene codes for multiple proteins that are translated independently

monocistronic

genes that produce mRNA coding for a single protein 

draw a polycistronic gene

x

operon

proteins coded on the same polycistronic gene that work towards the same goal

what happens when one promoter is turned on

allows bacteria to express multiple related proteins at once

when do genes get transcribed more frequently

when the product is in high demand

gene products only needed under certain circumstances

• availability of nutrients

• responding to threats

transcription factors

• proteins

• used in conjuction with RNA polymerases to provide further regulation

• regulate transcription by helping or hindering the interaction between RNA poly and promoter

transcription repressor

• proteins that decrease transcription of genes

• bind to the repressor binding site in the promoter

• physically prevents RNA polymerase from binding to it

example of negative regulation

transcription factor binding to promoter causing transcription repression

operator sequence

repressor binding site between -10 and -35

transcription activator

• proteins that increase gene transcription

• bind to an activator-binding site in the promoter

• physically helps RNA poly to bind to promoter

example of positive regulation

transcription factor binding to promoter causes transcription activator

B

activator binding site beside a weak promoter

how are transcription factors regulated

• can switch between an active and inactive state

• only bind to DNA when active

• can be regulated using post-translational modification

when are transcription factors regulated

• response to environmental conditions

• response to demanding survival conditions

small, organic molecules

• transcription factors are turned on/off by the presence/absence of one

• bind to binding pockets in transcription factors

• alter the shape of transcription factors

small organic molecules ex

• tryptophan

• allolactose

• cAMP

tryptophan operon

• codes for 5 proteins used to synthesize tryptophan

• E. coli controls the operon based on the concentration of trp in the cytoplasm

how does trp operon work

• low trp: turn on trp operon to produce more trp

• high trp: turn off trp operon to stop producing trp

trp operon promoter

• has -10 and -35 elements

• operator sequence for trpR binding in between -10 and -35

trp repressor

• TrpR

• negative regulator for trp operon

• not part of the trp operon

• expressed from a seperated monocistronic gene

• has binding pockets for aa trp

how is the trp repressor activated

• when trp binds to it

• activated trpR binds to the operator as a homodimer

why is trp operon an example of negative regulation

• trp binding to the trpR stops the production of trp

• activated trpR binds to the operator sequence, physically blocking the RNA poly holoenzyme from binding and producing more trp

2 types of negative regulation

• turn on repressor in the presence of a small molecule

• turn on repressor in the absence of a small molecule

turn on repressor in the presence of a small molecule 

• repressor becomes inactive when the molecule is removed

• ex. tryptophan repressor

turn on repressor in the absence of a small molecule

• repressor becomes inactive when a small molecule binds to it

• lac repressor

2 types of positive regulation

• turn on activator in the presence of small molecule

• turn on activator in the absence of small molecule

turn on activator in the presence of small molecule

• activator becomes inactive when the molecule is removed

• catabolite activator protein, CAP

turn on activator in the absence of small molecule

activator becomes inactive in the presence of a small molecule

what does e.coli use as a source of energy

• mostly glucose

• lactose

lactose

• disaccharide

• made of galactose and glucose

lactose metabolism

• digests lactose into galactose and glucose

• glucose enters glycolysis, krebs, etc

what does lactose metabolism use its extra energy for

• converts lactose into substrates of glycolysis

• does not occur if glucose is already available

what happens when theres high lactose and high glucose

glucose metabolism

what happens when theres low lactose and high glucose

glucose metabolism

what happens when theres high lactose and low glucose

lactose metabolism

what happens when theres low lactose and low glucose

uses other sugars

lac operon

codes for 3 proteins used to metabolize lactose into glucose and galactose

3 genes on lac operon

• lacA

• lacY

• lacZ

lacA

• codes for acetyltransferase

• physiological function of this enzyme is not well known

lacZ

• codes for beta-galactosidase

• hydrolyzes lactose to glucose and galactose

• 50% chance to produce allolactose as an intermediate molecule

lacY

• codes for lactose permease

• transports lactose in the environment into cytoplasm

• co-transports 1 H+ into cytoplasm

• this provides energy for the lactose transport

lac operon promoter composition

• weaker version of bacterial promoter

• weaker -10 and -35 elements

• UP element is absent

operators for the lac repressor

• operator 1

• CAP 

• operator 3

draw lac operon

x

what happens when lactose is low

• Lacl is expressed from another gene

• binds to operators

• bends the DNA in a loop

• makes it inaccessible for RNA poly holoenzyme

• no transcription from lac operon

when is Lacl active

in the absence of a small molecule

where does Lacl bind on the lac operon

• operator 3 and 1 OR

• operator 1 and 2

what shape is formed when Lacl binds to operators 3 and 1

homotetramer

background expression and ex.

• even when a strong repressor is active, very small amounts of the gene still gets transcribed

• no molecular mechanism is 100% efficient

• lacZ and lacY produced even in the presence of Lacl

allolactose

represses Lacl

why does e.coli have some lacY and lacZ at all times

background expression

what happens when lactose is available in the environment

• lactose gets transported into cell 

• some get converted into allolactose

• Lacl falls off DNA to make the promoter available for RNA poly holoenzyme to bind

what does lactose get converted to

• 50% - allolactose

• 50% - glucose + galactose

CAP

• activates lac operon

• helps RNA poly holoenzyme bind to weak promoter

when is CAP active

when cAMP is bound to it

adenylyl cyclase

converts ATP into cAMP

what happens when environmental glucose is high

• E coli does not want to activate lactose metabolism

• inhibits enzymatic activity

• cAMP low

• CAP remains inactive

when is adenylyl cyclase inhibited

when glucose gets imported from the environment

what happens when environmental glucose is low

• adenylyl cyclase becomes active

• produces cAMP

how does cAMP activate CAP

• 2 molecules of cAMP binds to CAP

• CAP binds to CAP binding site

• helps RNA poly holoenzyme bind

• activates transcription

CAP shape

• homodimer

• each monomer binds to 1 cAMP

• cAMP in the middle of the protein

• one CAP monomer directly makes contact with RNA poly holoenzyme

what controls lac operon

• Lacl

• CAP

draw response to lactose

x

draw response to glucose

x slide 35

when is transcription from lac operon turned on

• when lactose is high

• glucose is low

cytoplasmic glucose

• produced by lactose metabolism

• does not inhibit lac operon

what happens when environmental glucose and lactose is high

• adenylyl cyclase is inhibited

• CAP remains inactive

• lac operon promoter is open but does not transcribe

• E coli uses environmental glucose for energy

what happens when both environmental glucose and lactose are low

• Lacl binds to lac operon promoter

• CAP activated but cannot activate lac operon transcription since promoter is unavailable

• E coli uses a different sugar

transcription factors

• proteins that are expressed from their own gene

• expression may be regulated by other transcription factors

• regulate multiple promoters

• can alter gene expression

what do regulatory networks do

cause a cascade of transcription factors/repressors regulating themselves

how are networks regulated

• various environmental factors

• small molecules trigger specific responses

how many genes does TrpR repress

• 5

• one of them is the trpR gene, the gene that codes for trpR

TrpR autoregulation

• negative feedback loop

• stops making more TrpR when trp is high

• prevents over-repression of the trp operon to make it easier to turn it on once trp becomes low

how many genes does CAP control

• 180

• in response to glucose availability

CAP processes

• matabolism of carbon sources

• iron uptake

glucose catabolite repression

high environmental glucose downregulates processes carried out via CAP