Lecture #8

Constitutively expressed genes

Always expresses

Regulated genes

Expressed only when needed, induced or repressed

Transcriptional regulation

Involves turning on/off genes at the level of RNA synthesis

Post-transcriptional regulation

Involves steps after RNA is made

Susceptibility of regulation through access to DNA by RNA polymerase

Not a large issue in bacteria, more significant in eukaryotes

Susceptibility of regulation through recognition of promoter

Mostly regulated by sigma factors in bacteria

Susceptibility of regulation through initiation

Even if sigma-RNA pol recognizes the promoter, sometimes other proteins are needed to enhance initiation. Other proteins may block transcription from ignition to elongation

Susceptibility of regulation through elongation

Some proteins slow down elongation, or cause premature termination

Susceptibility of regulation through termination

Sometimes termination can be overridden by anti-terminator protein

How might promoter sequence impact RNA polymerase?

Different promoters have different strengths

These sequences bind sigma factors

Overriding Attenuation of trp operon

trp operon: a cluster of genes in bacteria that encode enzymes involved in the biosynthesis of the amino acid tryptophan

• attenuation occurs as long as tryptophan is plentiful

• If tryptophan is low, ribosomes stall, preventing the formation of a hairpin, where there is typically attenuation

Cis-acting elements

DNA sequences located near a gene that directly regulate its expression

trans-acting elements

DNA sequences encoding upstream regulators (ie. trans-acting factors), which may modify or regulate the expression of distant genes. Trans-acting factors interact with cis-regulatory elements to regulate gene expression.

Lac operon

• first operon discover

• lacY: Galactoside permease (transports lactose into cells)

• lacZ: B-galactosidase (cuts lactose into galactose and glucose)

• lacA: Galactoside transacetylase (function unclear)

  • all 3 genes transcribed together producing 1 mRNA

  • has its own ribosome binding site

  • Can be translated by separate ribosomes that bind independently of each other

Diauxic growth of E. coli

two-phase growth pattern that occurs when microorganisms are given two sources to metabolize

• cells are grown in both glucose and lactose

Lactose

Disaccharide, galactose and glucose joint by a b-galactosidic bond

• needs to be imported into E. coli and digested into a monosaccharide

Negative Control of the lac operon

Lac repressor must be removed

• where a repressor protein, encoded by the lacI gene, binds to the operator region on the DNA, preventing transcription of the lac operon genes unless lactose (the inducer) is present

• Minor metabolic product of lactose, allolactose, is the inducer, removing tetramer and allowing transcription of b-galactosidase to break down lactose

IPTG (Isopropylthiogalactoside)

Analog for Allolactose (inducer), but cannot be broken down as an energy source, so it keeps lac operon “on”

• colonies that are on are blue because it is producing b-galactosidase

Positive controller of lac operon

• cAMP

• A protein factor:

  • Catabolite activator protein (CAP)

  • Cyclic-AMP receptor protein (CRP)

  • gene encoding this protein is crp

Catabolite activator protein

• cAMP added to E. coli can over come catabolite repression of the lac operon

• addition of cAMP leads to activation of the lac gene even in the presence of glucose

CAP-cAMP complex

Positively controls activity of b-galactosidase

• CAP binds to cAMP tightly

• mutant CAP not bind cAMP tightly

• Compare activity and production of B-galactosidase using both complex

• low activity with mutant CAP-cAMP

How does CAP-cAMP complex positively regulate lac operon

CAP-cAMP complex allows the formation of the open promoter complex for the RNA polymerase

proposed:

• CAP-cAMP dimer binds to its target site on the DNA

• alpha CTD of polymerase interacts with the specific site on CAP

• Binding is strengthened between promoter and polymerase

ara operon use

Coded for enzymes required to metabolize the sugar arabinose

• catabolite-repressible operon

Structure of ara operon

• 3 genes (araB, A, and D) encode for arabinose metabolizing enzymes

  • transcribed rightward from promoter araPbad

• Other gene, araC

  • encodes the control protein AraC

  • Transcribed left ward from the araPc promoter

Control of ara Operon

When lacking arabinose, a loop is formed that blocks binding site

• RNA polymerase just bounces off

When there is arabinose, binding to araPc is able to occur and transcription of araBAD can occur

trp operon

Contains the gene for the anabolic enzymes (build up a substance) the bacterium needs to make the amino acid tryptophan

• turned off by a high level of the substance provided

• operon is subject to negative control by a repressor when tryptophan levels are elevated

negative control of trp operon

5 genes code for polypeptides in the enzymes of tryptophan synthesis

• high tryptophan concentration signals to turn off the operon

• the presence of tryptophan helps the trp repressor bind to its operator

No tryptophan: No trp repressor, just inactive protein aporepressor

If aporepressor binds tryptophan, changes conformation with high affinity for trp operator → forms trp repressor→ tryptophan is a corepressor

Riboswitches

Small molecules can act directly on the 5’ UTRs of mRNAs to control their expression