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Biochem 385 Unit 3 > Lecture 37: Prokaryotic Gene Regulation > Flashcards

Lecture 37: Prokaryotic Gene Regulation Flashcards

1
Q

primary determinant of protein expression

A

DNA Transcription… aka

GENE REGULATION

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2
Q

basic gene unit in prokaryotes

A

operons

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3
Q

what does operon include

A

gene regulatory sequences (operator)
promoter
coding sequences

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4
Q

what do operons allow for

A

coordinate expression of regulated genes

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5
Q

lac operon regulated by

A 
repressor protein (lac repressor)
cAMP receptor protein (CRP)-- activator protein

both bind to lac operon DNA
both DNA binding proteins

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6
Q

trp operon regulated by

in E. coli

A

1) transcriptional repression

2) transcriptional attenuation mediated by ribosome pausing

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7
Q

why attenuate?

A

because transcription and translation are happening at same place and time

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8
Q

trp operon regulated by

in Bacillus

A

TRAP protein

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9
Q

levels of protein synth regulation

A

after transcription and synth of mRNA can be modulated by….

RNA turn over
rates of protein synth and turnover

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10
Q

long term mech of control is…

A

DNA transctiption (gene regulation)

if you dont need the protein product, don’t start down path of making it

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11
Q

prokaryote operon units

A

genes to be transcripbed
promoter region
1 or more regulatory sequence

allows coordinated expression of genes

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12
Q

transcription factors

A

DNA binding proteins
bind directly to DNA to regulate
binding by small molecs affects structure of proteins, which affects their DNA binding affinity

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13
Q

Positive and Negative Gene Regulation:

Repressors

A

Two ways:
1) Repressor bound to DNA w/o ligand is off=no TRX
ligand conc builds up, binds to repressor, repressor dissociates and mRNA can be transcribed
2) repressor bound to DNA W/ ligad= no transcription
ligand conc decs, ligand falls off, repressor protein dissociates, transcription happens

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14
Q

so what does the ligand conc do?

A

regulates binding events

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15
Q

Constititively

A

state the thing is in

the default state

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16
Q

Positive and Negative Gene Regulation:

Activators

A

1) protein bound to ligand, protein is ON, activates TXN, when ligand falls off, protein dissociates from DNA, txn less
2) when no ligand, protein is on, activates TXN. when ligand binds, protein dissociates, TXN less

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17
Q

How do represors and activators affect RNA Pol activity?

A

1) repressors sterically (physically) block binding of RNA pol
2) activators work molecularly to help bring things together. Protein protein interaction

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18
Q

Activator proteins and repressors can both…

A

be regulated by ligand binding

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19
Q

Lac operon encodes…

A

genes that will be involved in lactose metabolism

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20
Q

what is preferred carbon soruce in e. coli?

A

glucose

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21
Q

what happens when glucose is low?

A

we use lactose (if available) for metabolization

lac operon needed to provide the enzymes needed for lactose metabolism

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22
Q

lactose permease

A

imports lactose

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23
Q

beta-galactosidase

A

cleaves lactose into to glucose and galactose

synths low levels of allolactose (a lactose metabolite)

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24
Q

allolactase

A

inducer molecule for the lac repressor
when it binds to the repressor, it inactivates the repressor
thus turning operon on

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25
two things needed to metabolize lactose
lactose permease beta-galactosidase these are part of lac operon
26
Parts of lac operon
promoter region regulatory region operator region 3 genes that encode proteins
27
3 genes that encode proteins
lacZ lacY lacA
28
lacZ gene
makes beta-galactosidase
29
lacY gene
lactose permease
30
lacA gene
transacetylase (not needed for lactose metabolism)
31
is lac operon usually on or off?
OFF (because we usually use glucose, not lactose) | but it is induceable if theres lots of lactose and no glucose
32
how to regulate lac operon?
binding of lac repressor | and lac operator
33
where does the lac repressor come from?
its around encoded by nearby DNA, but not part of operon it is ALWAYS around, its activity depends on whether inducer molec is around
34
what is the inducer molec for the lac repressor
allolactase when theres enough around, it binds to repressor, inactivates it, and turns operon on
35
high glucose, low lactose
operon off lac repressor is on: binds to promoter, prevents RNA pol from binding and making lacY and lacZ genes TRNASCRIPTION OFF
36
glucose and lactose high
allolactose made, binds to repressor and inactivates it (b./c lactose high) BUT NOT TNX b/c CRP is not bound to DNA (b/c glucose high) glucose is preferred! TXN off (pretty much)
37
CRP
activator protein needed to turn operon on is inactive if glucose high only active when glucose low, lactose high
38
where can lac repressor bind?
at multiple operator sites(3) | O1 and O2 (these play major role in repression)
39
lac repressor binding
tetramere forms DNA-protein interaction loops DNA out to prevent RNA pol from binding
40
when allolactose bound to repressor
HUGE conf change | this dramatically decreases DNA binding affinity
41
when glucose is low and lactose is high
low glucose=cAMP levels rise, bind to CRP, CRP activates DNA binding activity high lactose= allolactose binding to repressor active activator, inactive repressor TRANSCRIPTION ON
42
active activator helps...
RNA pol bind and transcribe genes needed to metabolize lactose
43
what does cAMP do?
binds to CRP turns activator on (txn on) binding to CRP bends the DNA
44
what is adenylate cyclase activated by? What does it do?
low glucose it makes cAMP its a bacterial thing with a glucose binding domain
45
Slide 11 for review
Slide 11 for review
46
explain why adding glucose to a bacterial culture containing lactose inhibits beta-galactose expression
because transcription is off if glucose levels are high
47
what happens of both glucose and lactose are low?
transcription is off both activator and repressor are bound, but repressor wins see experiment, almost new beta galactosidease is made
48
what happens of glucose is low and lactose is high?
txn is on
49
what happens if glucose and lactose are both high?
txn is off
50
regulation of E. coli trp operon
1) transcriptional repression | 2) transcriptional attenuation by ribosome stalling in mRNA
51
how many amino acids needed to make tryptophan
5-6 depending on organism
52
why would E. coli use TWO mechanism to regulate trp biosynthesis?
to make sure its really really off so we don't waste energy | there are 5 enzymes involved! It takes a lot of nrg to make these amino acids! Holy cow!
53
trp operon
promoter operator leader sequence 5 genes that make the 5 enzymes
54
trp repressor...
always being made inactive in absence of tryptophan works as repressor when there IS tryptophan only make tryptophan if there is none
55
where does trp repressor bind?
at operator site
56
trp repressor only active repressor...
when bound to tryptophan! | when it is bound to tryp, it will bind to DNA and block transcription
57
leader sequence
used with the attenuation mech part of regulatory sequence comes before the genes for txn 4 regions
58
trp repressor
works just like the lac repressor pretty much | inactive when there is no tryp (so transcirption can happen)
59
``` transcriptional attenuation: 4 regions of leader sequence region 1 (lots of tryptophan) ```
encoders for tryptophan | when ribosome gets here, if there is enough tryp around, RNA pol just keeps synthing
60
``` transcriptional attenuation: 4 regions of leader sequence region 2 (lots of tryptophan) ```
if there is enough tryp around, the ribosome gets here quickly Blocakge of sequence 2 before it can base pair with sequence 3
61
transcriptional attenuation: 4 regions of leader sequence | region 3 and 4 (lots of tryptophan)
if ribosome is going fast, then 3 and 4 have to base pair, | hairpin structure between them forms
62
what does formation of 3-4 hairpin structure do?
causes RNA Pol to fall off so transcription of entire operon doesn't happen does not continue past leader sequence we don't make more tryptophan!
63
transcriptional attenuation: when tryptophan levels are low
ribosome has to wait for a charged tRNA w/ tryptophan on it ribosome goes slowly, then stalls out while waiting, RNA pol can get a head start. ribosome doesn't cover up sequence 2, so 2-3 can basepair then 2-3 base pair (not really well) (doesnt make RNA pol stop) RNA pol is not stopped, tryptophan can be made
64
2-3 base pair
DOES NOT act as attenuator
65
Regulation of Bacillus trp operon
DOES NOT USE trp repressor protein or ribosome stalling uses large RNA binding protein repeated sequences in region upstream of genes
66
what does Bacillus use of large RNA binding protein accomplish?
binds to tryptophan to halt transcription when trp levels are high
67
Bacillus if lots of trp
tryptophan binding to TRAP (repeated protein that looks weird, binds to repeating RNA structure) formation of hairpin structure txn stops
68
Bacillus if little trp
when transcribed to mRNA, normal structure forms, pol can read through region, txn happens
69
TRAP binding (bacillus)
induces RNA hairpin to terminate transcription
70
what might explain the evolution of yet another trp regulatory mechanism?
independent origins? making unneeded gene products is really expensive really need to make sure we don't make what we don't need

Biochem 385 Unit 3 (11 decks)

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