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Homework

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• Two names did not register• R10780801• R11275224

Exam 3

• Constitutive transcription – continuous expression usually for genes that perform routine tasks necessary for life

• Regulated transcription – expression at particular times for genes that are differentially required under varied conditions

• Regulated transcription includes control of both initiation and amount of transcription

• Control is modulated by interactions between proteins and regulatory sequences within the DNA

• Negative control – binding of a molecule to prevent transcription• Works via repressor proteins

• Positive control – binding of a molecule to encourage/initiate transcription• Works via activator proteins

Bacterial Gene Regulation

• Protein-nucleic acid binding• Most proteins to be discussed bind

specific DNA/RNA sequences• Most commonly via α-helix insertion

into major groove(s)

Bacterial Gene Regulation

• Protein domains - Regions of a protein that have a particular function

• DNA binding domains have amino acids that associate with nucleotides of particular DNA sdquences

• Specificity is dictated by the unique patterns of atoms in these nucleotides

• Most commonly via α-helix insertion into major groove(s)

Bacterial Gene Regulation

• Protein domains - Regions of a protein that have a particular function

• Most regulatory proteins have at least two domains• DNA binding• Allosteric

• Often have a third• Multimerization

Bacterial Gene Regulation

• Allosteric regulation• Protein shape is intimately related

to function• Molecule binding can significantly

alter shape• Ligand – a molecule binding to a

complementary site on a protein to alter its conformation

• Allostery – “other shape”• A ligand binds and the protein

changes shape to reveal or conceal another binding site

Bacterial Gene Regulation

• Allosteric regulation• Small molecule effectors• http://bio156.aznetwork.com/

animated!/chapter04/videos_animations/allosteric_inhibition.html

Bacterial Gene Regulation

• Negative control • Two scenarios

• Repressor protein has an active DNA-binding domain in the absence of an inducer ligand

• Repressor protein has an inactive DNA-binding domain in the absence of a co-repressor

Bacterial Gene Regulation

• Positive control • Two scenarios

• Activator protein has an inactive DNA-binding domain in the absence of an effector ligand

• Repressor protein has an active DNA-binding domain in the absence of a inhibitor

Bacterial Gene Regulation

• The lac operon• Operon – a cluster of genes undergoing coordinated regulation• Very common in bacteria• The lactose (lac) operon is responsible for producing three

polypeptides responsible for the metabolism of lactose• Glucose is the preferred energy source

• metabolized via glycolysis• a monosaccharide

• Lactose is an alternative, only metabolized if needed• A disaccharide

• Thus, the lac operon is inducible, turned on when needed but off the rest of the time

Bacterial Gene Regulation

• The lac operon• One gene (lacZ) in the lac operon encodes

-galactosidase, which breaks the bond between galactose and glucose in the lactose molecule

• Glucose and galactose can then be metabolized via glycolysis

• One intermediate product is allolactose

Bacterial Gene Regulation

• The lac operon structure• A multi-part regulatory region and three structural genes

• Structural genes• LacZ -galactosidase• LacY permease• LacA transacetylase

• One other gene, LacI (located upstream, not part of the operon), encodes the lac repressor protein

Bacterial Gene Regulation

• Another gene, LacI (located upstream), encodes the lac repressor protein

• Three domains• DNA binding• Multimerization• Allosteric

• Forms a homotetramer• Allolactose is the ligand that induces

conformational change decreased DNA binding

Bacterial Gene Regulation

• One last gene encodes catabolite activator protein (CAP)• Three domains

• DNA binding• Multimerization• Allosteric

• Forms a homodimer• Cyclic AMP (cAMP) is the ligand that induces conformational change

increased DNA binding• cAMP is produced only when glucose is not present

Bacterial Gene Regulation

• The lac operon structure• A multi-part regulatory region

• CAP binding site – bound by catabolite activator protein• Promoter – bound by RNA polymerase• Operator – bound by lac repressor

Bacterial Gene Regulation

• Let’s put it all together• Three scenarios exist

• Lactose and glucose present

• Glucose +, lactose –

• Lactose +, glucose -

Bacterial Gene Regulation

What does the cell ‘want’ to do in each case and how is it accomplished?

• Glucose +, lactose =(normal metabolism)• The cell ‘wants’: to use glucose, no lactose is available so why

bother transcribing genes to metabolize it?• The cell accomplishes this by: shutting down the lac operon

Bacterial Gene Regulation

Negative controlNo allolactose present repressor is active Repressor binds to operator blocks transcription

• Glucose -, lactose + =(lactose metabolism)• The cell ‘wants’: to use glucose but there isn’t any, lactose is

available so it must transcribe genes to metabolize it• The cell accomplishes this by: activating the lac operon

Bacterial Gene Regulation

Positive controlAllolactose present repressor is inactiveRepressor cannot bind to operator transcriptionCAP binds CAP binding site recruits RNA pol, increasing transcription

• Glucose +, lactose +• The cell ‘wants’: to use both but no need to expend large amounts

of extra energy by specially targeting lactose for use• The cell accomplishes this by: mostly metabolizing glucose but

allowing the lac operon to be transcribed at a minimal level

Bacterial Gene Regulation

Allolactose present repressor is inactive, transcription can happenGlucose is present no cAMP no CAP binding no RNA pol recruitment

minimal lacZ transcription

https://www.youtube.com/watch?v=mwkI5VFd1Gg - Watch just for the musichttp://highered.mheducation.com/olc/dl/120080/bio27.swf

• Wait a minute?!?!• If no lactose is present, transcription is shut down• No permease is available to allow lactose in• And even if it got in, it wouldn’t be metabolized and no allolactose

would be produced to release the repressor from the operator• How does transcription EVER start?

• Leaky transcription• Binding of the repressor is reversible• Sometimes it just falls off, allowing a very low level of transcription

and low levels of permease and -galactosidase in the cell

Bacterial Gene Regulation

• The trp operon• Tryptophan is an essential amino acid that can be synthesized by

the cell• But, why bother if tryptophan is already present?• The trp operon is repressible, meaning it’s usually on but can be

turned off• Furthermore, it can be fine tuned to match the needs of the cell a

process called attenuation

Bacterial Gene Regulation

Bacterial Gene Regulation• The trp operon structure• A multi-part regulatory region and five structural genes

• Structural genes• trpA-E enzymes involved in the anabolism (building

molecules• trpR elsewhere, encodes trp Repressor

• trp repressor protein has a similar structure but works the opposite way of lac repressor

• Three domains• DNA binding• Multimerization• Allosteric

• Forms a homodimer• Tryptophan is the ligand (corepressor) that

induces conformational change increased DNA binding

Bacterial Gene Regulation

• The lac operon structure• A multi-part regulatory region

• Promoter – bound by RNA polymerase• Operator – bound by trp repressor• Attenuator – we’ll get to that

Bacterial Gene Regulation

• Let’s put it all together• Three scenarios exist

• Tryptophan + as needed

• Tryptophan + but low

• Tryptophan -

Bacterial Gene Regulation

What does the cell ‘want’ to do in each case and how is it accomplished?

• Tryptophan + • The cell ‘wants’: to use use the available tryptophan, so why bother

transcribing genes to metabolize it?• The cell accomplishes this by: shutting down the trp operon

Bacterial Gene Regulation

• Tryptophan - • The cell ‘wants’: tryptophan and needs to manufacture it for itself• The cell accomplishes this by: activating the trp operon

Bacterial Gene Regulation

• Tryptophan +/-• The cell ‘wants’: some tryptophan but not too much fine tune

production• The cell accomplishes this by: attenuating (taper off) the trp operon• As tryptophan increases in the cell production decreases• As tryptophan decreases in the cell production increases• The result is a steady-state, or homeostasis• Attenuation involves the leader strand (trpL) segment of the trp

operon mRNA• Somehow, increased tryptophan availability results in the premature

termination of trp operon transcription

Bacterial Gene Regulation

• Tryptophan +/-• As trp increases rate of trp operon transcription decreases• Of the transcripts that are produced, more and more consist only of

the first 140 nt from the 5’ end of trpL

Bacterial Gene Regulation

trp =

Full length transcripts

Partial (inviable) transcripts

• Tryptophan +/-• trpL contains• Four repeated DNA sequences

• Can form stem-loop structures• A region that codes for a 14 AA polypeptide

• Two back-to-back codons code for tryptophan

Bacterial Gene Regulation

• Tryptophan +/-• trpL contains• Four repeated DNA sequences

• Can form stem-loop structures• A region that codes for a 14 AA polypeptide

• Two back-to-back codons code for tryptophan

Bacterial Gene Regulation

• Tryptophan +/-• The four repeats can form three, mutually exclusive structures• 2-3 loop = antitermination loop• 3-4 loop = termination loop

Bacterial Gene Regulation

• Tryptophan +/-• 3-4 loop = termination loop• Remember WAY back in chapter 8?• Intrinsic termination• If 3-4 loop forms, transcription of trp is stopped

Bacterial Gene Regulation

• Tryptophan +/-• 2-3 loop = antitermination loop• 3-4 loop cannot form transcription continues• How does the cell control this in such a way to encourage or

discourage trp expression?• As tryptophan increases in the cell transcription decreases• As tryptophan decreases in the cell transcription increases

Bacterial Gene Regulation

• Tryptophan +/-• How does the cell control this in such a way to encourage or

discourage trp expression?• Depends on whether or not the ribosome is stalled in region 1• If tryptophan is readily available, ribosome has no trouble filling the

need for two sequential tryptophans during translation• Ribosome moves rapidly through 1 and covers 2, preventing 2 from

interacting with 3• 3-4 termination loop forms, halting transcription of the full operon

Bacterial Gene Regulation

• Tryptophan +/-• If tryptophan is readily available, ribosome has no trouble filling

the need for two sequential tryptophans during translation• Ribosome moves rapidly through 1 and covers 2, preventing 2

from interacting with 3• 3-4 termination loop forms, halting transcription of the full operon

Bacterial Gene Regulation

• Tryptophan +/-• How does the cell control this in such a way to encourage or

discourage trp expression?• Depends on whether or not the ribosome is stalled in region 1• If tryptophan is in short supply, ribosome has difficulty filling the need

for two sequential tryptophans during translation• Ribosome stalls at 1, allowing 2 to interact with 3• 2-3 antitermination loop forms, allowing transcription of the full operon

Bacterial Gene Regulation

• Tryptophan +/-• If tryptophan is in short supply, ribosome has difficulty filling the

need for two sequential tryptophans during translation• Ribosome stalls at 1, allowing 2 to interact with 3• 2-3 antitermination loop forms, allowing transcription of the full

operon

Bacterial Gene Regulation

• Tryptophan +/-• http://highered.mheducation.com/sites/dl/free/

0072835125/126997/animation28.html

Bacterial Gene Regulation