Transcript Bacterial Gene Regulation

Homework
• Active from 3:20 today thru midnight on November 25
Exam 3
• 37 valid questions
• Average score – 76.3%
Letter grade distribution
120
100
80
• Two names did not
register
• R10780801
• R11275224
60
40
20
0
A
B
C
D
F
Bacterial Gene Regulation
• 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/animat
ed!/chapter04/videos_animations/al
losteric_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 -
What does the cell ‘want’ to
do in each case and how
is it accomplished?
Bacterial Gene Regulation
• 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
Negative control
No allolactose present  repressor is active 
Repressor binds to operator  blocks transcription
Bacterial Gene Regulation
• 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
Positive control
Allolactose present  repressor is inactive
Repressor cannot bind to operator  transcription
CAP binds CAP binding site  recruits RNA pol, increasing transcription
Bacterial Gene Regulation
• 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
Allolactose present  repressor is inactive, transcription can happen
Glucose is present  no cAMP  no CAP binding  no RNA pol recruitment 
minimal lacZ transcription
http://highered.mheducation.com/olc/dl/120080/bio27.swf
https://www.youtube.com/watch?v=mwkI5VFd1Gg - Watch just for the music
Bacterial Gene Regulation
•
•
•
•
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
• 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
Bacterial Gene Regulation
• 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 -
What does the cell ‘want’ to
do in each case and how
is it accomplished?
Bacterial Gene Regulation
• 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
Partial (inviable) transcripts
trp =
Full length transcripts
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 +/• 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