lec 9.3 - gene expression in bacteria, examples (copy)

EX: Lactose operon

• definition

• types of control

• Group of genes that produce enzymes that allow E.coli to use lactose as a carbon source

• controlled by both +ve and -ve control

  • +ve: turns expression on

  • -ve: turns off transcription

describe the make up of the lac operon

• the genes

• promoters

• the mRNA made

• The lac operon occupies ~6,000 bp of DNA

• The lacI gene has its own promoter and terminator (therefore makes its own mRNA and protein). The end of the lacI region is adjacent to the lacZYA promoter, P (one promoter for 3 genes → makes polycistronic mRNA)

• The transcription from P produces one polycistronic mRNA that has 3 translational reading frames that produce proteins from the 3 genes, lacZ, lacY and lacA genes.

Lactose operon – 3 parts

• Part 1 contains operon with 3 genes, lac Z, lac Y and lac A with one promoter and one operator sequence

• Part 2 is gene (lac I) that produces the repressor protein, not regulated (always on, always repressed → under negative control)

• Part 3 is a common gene that produces an activator protein

describe Lac Y and Lac Z genes products

• Lac Y gene – produces galactoside permease

• Lac Z gene – produces βgalactosidase –

  • isomerizes lactose and produces allolactose - used as inducer

  • can also cleave lactose into galactose and glucose - used as carbon sources

How the operon works - Negative regulation

to turn ON transcription → need inducer → causes induction

• inducer binds to repressor protein to change its shape so it can’t bind to the operator

• this allows RNA pol to bind to the promoter instead

• negative regulation to inactivate the repressor

What does repressor look like?

• The repressor tetramer consists of two dimers.

• Dimers are held together by contacts involving core subdomains 1 and 2 as well as by the tetramerization helix.

• The dimers are linked into the tetramer by the tetramerization interface

inducer binding to repressor consequences

• The inducer changes the structure of the core so that the headpieces of a repressor dimer are no longer in an orientation with high affinity for the operator

How does repressor interact with operator sequence?

• Repressor is tetramer

  • One dimer attaches via a helix-turn-helix to major groove and can then bind to other dimers to give tetramer that reduces expression of genes

  • presence of O2 alone is enough to repress transcription

How the operon works - Positive regulation

• CAP protein or CRP protein (catabolite regulator protein), a trans-activator protein

• Activated by binding cAMP (inducer)

• If cAMP not available all sugar operons are turned off – called catabolite repression

  • regulated by level of glucose (increase in glucose and ATP levels turn off the operon because that means it is not needed)

How does Glucose regulate the level of cAMP?

• by inhibiting enzyme that makes cAMP

ORI and CRP sites

• describe them

• ORI sites bind to repressor (stops RNA polymerase from binding to promoter)

• CRP site binds to catabolite regulator protein (helps RNA polymerase bind to promoter to turn on transcription), needs cAMP to be active, turns on transcription

when glucose high, cAMP low, lactose absent

when there's plenty of glucose, low cAMP, and no lactose to break down, the cell conserves energy by keeping the lac operon turned off

• lacI promoter binds RNA pol so the repressor is made

• the repressor binds to the lacZYA promoter so these genes are not expressed

when glucose low, cAMP high, lactose present

• when there's low glucose, high cAMP, and lactose is present, the cell senses the need for an alternative energy source

• Allolactose, a byproduct of lactose metabolism, binds to the repressor, allowing the lac operon to be activated

  • enables the cell to produce the enzymes necessary for breaking down lactose

when glucose low, cAMP high, lactose absent

• when there's low glucose, high cAMP, but no lactose to metabolize, the lac operon remains off

• absence of lactose means there is no need for the cell to activate the lac operon and produce enzymes for lactose metabolism

• repressor protein continues to block the expression of the lac operon, conserving energy for the cell

  • repressor stays on because there is no allactose to bind to it

when glucose high, cAMP low, lactose present

• in the presence of high glucose, low cAMP, and lactose (with allolactose), the lac operon is not fully induced

• repression from the lack of cAMP-CAP complex prevents high-level gene expression, and the lac operon operates at a reduced level

• allows the cell to prioritize using glucose while still being prepared to use lactose when needed

Mutant in repressor gene

• no repressor protein produced

• effect: basal transcription

Mutant in operator sequence

• repressor has nothing to bind to

• effect: no repressor to repress so transcription happens

mutation in Lac Z gene?, Lac Y gene? Promoter sequence? Activator sequence?

• Lac Z: still have expression

• Lac Y: can’t get lactose into cell

• promoter sequence: no RNA pol binding → no transcription

• activator sequence: no CAP protein binding, basal transcription

You produce a F’ strain. The resultant strain is partial diploid for the lactose operon. You now generate mutants of the operon genes and get the following results and grow them in media with no glucose and with and without lactose. Fill in the table.

see image

How can we study diploid organisms? (the effect of each allele on the phenotype?)

• We can make partial diploids in bacteria

  • Partial diploids (merodiploids) have 2 copies of the genes of interest – one copy on the chromosome and one copy on a plasmid

Artificial inducers

• examples

• Artificial inducers can interact with repressor and allow operon to be transcribed

• They are not used up by the enzyme of the operon and so continue to induce the operon

• X-gal - substrate for Beta-galactosidase, turns blue when digested

• IPTG- artificial inducer: looks like allolactose enough to act as an inducer