Midterm 3 Lecture 3: Prokaryotic gene expression

when is the lac operon “on”

when lactose level is high and glucose is low

what is a promoter

a region of dna (dna sequence) that signals to rna polymerase where to begin transcription (where rna pol binds)

what is an activator

protein that directly binds to DNA to inc binding / activity of RNA pol

what is a repressor

protein that binds to operator to block binding of rna pol

what is an effector

umbrella term for small molecules that modulate activators/repressors

what are inducers

specific type of effector → small molecule that either binds to repressors and inhibits them or binds to activators and activates them

where are promoters typically located

upstream of the start site

t/f: all genes are transcribed at the same time

false →

what determines when and how much mRNA is made in prokaryotes

the promoter and surrounding sequences

• binding of proteins to these regions of dna can inhibit or enhance transcription (eg in lac operon in e. coli)

t/f: gene expression is only regulated at transcription

false → regulated at many levels

explain transcriptional control of gene regulation in eukaryotes

• binding of RNA pol to promoter

• shift from initiation to elongation

• release of mRNA at termination

what is an operon

a cluster of genes transcribed together

note: not all genes are a part of an operon → but genes not part of an operon can still be regulated

what is polyciotronic mRNA

mRNA that encodes for more than one protein

what is catabolism and what does it require, and is it off or on by default

breaking down larger molecules to obtain E and smaller molecules

• requires inducible regulation

  • pathway should be off by default and turned on only when the molecules to be broken down are present

what is anabolism and what does it require and is it off or on by default

use of smaller precursor molecules and E from catabolism to build larger molecules

• requires repressible regulation

  • pathway should be on by default unless cell has enough of the larger molecule

which 2 enzymes are required for lactose utilization

1. permease

   • transports lactose into the cell

2. beta-galactosidase

   • splits lactose into glucose and galactose

when there is no lactose present, how much permease and beta-galctosidase is produced

very little

t/f: the lac genes are transcribed together

true

what does the lac operon contain

lacZ, lacY, lacA, and regulatory elements (promoter and operator)

when lacZ expression is repressed or induced, which effects would be observed for lacY and lacA

the same effect

• lead to proposal that the 3 genes are transcribed as part of same polycistronic mRNA

explain the overall regulation of the lac operon when glucose is NOT present

is “off” by default (we only want it on when lactose is present)

• presence of lactose induces expression of the genes required for lactose utilization

• addition of lactose to growth medium induces a 1000 fold inc in the production of lac permease and b-galactosidase

what is the repressor protein in regards to the lac operon

has a specific sequence that binds to operator and inhibits transcription

• produced by gene lacI (which is not part of the lac operon)

• in absence of lactose, repressor binds to operator and prevents transcription (rna pol cant bind to promoter)

• lac repressor is a negative regulatory element

explain the structure of the lac repressor

forms an active tetramer (has 4 subunits)

• binds to two conserved sequences of dna

  • causes it to loop

• looping of dna prevents access of rna pol to promoter

explain the induction of the lac operon when lactose is present

when lactose is present…

• proportion of lactose is converted to allolactose by a b-galactosidase side rxn

• allolactose is the inducer

  • inducer binds to repressor and prevents repressor from binding to the operator

    • making the repressor an allosteric repressor

  • no repressor bound to operator means that rna pol can bind to the promoter and transcription can occur

what are allosteric repressors

• repressors that reversibly change shape when inducer is bound are allosteric repressors

how does lactose enter the cell and get converted to allolactose to induce transcription if the enzymes needed to do this aren’t being transcribed

operators are leaky

• they are never completely on or off

• repressor binds to operator but not covalently (can come off)

so basically without allolactose present, the more active the repressor is, the more binding it does w the operator, the less transcription that happens

note: there will always be some residual permease and beta-galactosidase in the cell

what is some evidence for a lac repressor protein (think lac-)

some mutations in lacl (lacl-) result in synthesis of b-galactosidase (lacZ) and permiase (lacY) even in the absence of lactose

• these are examples of constitutive mutants

what are constitutive mutants

• mutations that result in constitutive (always on) expression

• protein synthesis occurs regardless of environmental conditions

what is some more evidence for a lac repressor protein (think lacl^S)

lacl^S is a super repressor → can not be reduced (always bound to operator even with allolactose present → so nothing else can bind to the operator or promoter)

• results in NO synthesis of b-galactosidase (lacZ) and permease (lacY) even in the presence of lactose

what is a mutant repressor that cant bind an inducer called

a superrepressor

what is O^c or lacO^c

a mutant operator → c = constitutively on

• repressor cannot recognize and bind to operator, so lac enzymes are synthesized constitutively

note: both lacl- and Oc mutations result in constitutive expression of the lac operon

explain cis action and state what typically acts in cis

cis elements affect regulation of nearby genes on same DNA molecule

• DNA sites act in cis

explain trans action and state what typically acts in trans

trans elements can affect regulation on diff dna molecules

• proteins act in trans (can float around and affect things elsewhere)

what is a plasmid

a small (smaller than chrom) independently replicating segment of dna common in bacteria

what is transformation

bacteria can take up (actually take in and incorporate) small bits of dna (not from them, like a plasmid)

what are merodiploids

partial diploid cells

what is the cis/trans test

use plasmids to make partial diploid cells (merodiploids) to study the lac operon

• can add lactose with working gene that is fefective in the og cell and if there is a restoration of fx for the whole cell that means the proteins encoded for by the palsmid’s genes were able to travel to the og chromosome in the dna and effect it too → :. trans acting

is lacI a cis or trans acting element

trans → repressors can move

is o+ a cis or trans acting element

cis → it is an operator :. cant move

how many adults worldwide have lactose intolerance

68%

why do people have lactose intolerance

don’t produce enough lactase (basically same fx as b-galactosidase)

does the lac operon turn on if lactose and glucose are both present

no → it wont try and break down lactose for E if glucose is present

explain regulation of the lac operon in the presence of glucose

glucose is the preferred substrate of e. coli

• in absence of glucose, cAMP levels inc

  • cAMP production is inhibited by glucose

    • cAMP is an inducer

• cAMP binds to cAMP receptor protein (CRP)

  • cAMP receptor protein is an activator (positive regulator) of lac transcription that is active when bound to cAMP, it’s inducer

    • CRP binds to the dna and inc the efficiency of rna pol binding activity

what type of action does glucose work on to lower cAMP production

catabolite repression: the overall effect of glucose is to prevent lac gene expression

• anenylyl cyclase produces cAMP → glucose inhibits adenylyl cyclase

explain regulation by lactose

1. repressor protein

   • produced by lacI and binds to operator, blocking transcription (by blocking rna pol from binding to promoter)

2. Allolactose

• present when lactose is present

• is an inducer (type of effector)

• binds to repressor protein and prevents it from binding to operator

explain regulation by glucose

• when glucose is absent, cAMP levels are high

  • cAMP is inducer for the activator CRP

• cAMP-bound CRP is active and binds near promoter, inc rna pol binding/activity

• when glucose is present, cAMP is low, CRP will not bind to cAMP, activator is inactive