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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
Q

two things needed to metabolize lactose

A

lactose permease
beta-galactosidase

these are part of lac operon

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26
Q

Parts of lac operon

A

promoter region
regulatory region
operator region
3 genes that encode proteins

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27
Q

3 genes that encode proteins

A

lacZ
lacY
lacA

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28
Q

lacZ gene

A

makes beta-galactosidase

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29
Q

lacY gene

A

lactose permease

30
Q

lacA gene

A

transacetylase (not needed for lactose metabolism)

31
Q

is lac operon usually on or off?

A

OFF (because we usually use glucose, not lactose)

but it is induceable if theres lots of lactose and no glucose

32
Q

how to regulate lac operon?

A

binding of lac repressor

and lac operator

33
Q

where does the lac repressor come from?

A

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
Q

what is the inducer molec for the lac repressor

A

allolactase

when theres enough around, it binds to repressor, inactivates it, and turns operon on

35
Q

high glucose, low lactose

A

operon off
lac repressor is on: binds to promoter, prevents RNA pol from binding and making lacY and lacZ genes

TRNASCRIPTION OFF

36
Q

glucose and lactose high

A

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
Q

CRP

A

activator protein
needed to turn operon on
is inactive if glucose high
only active when glucose low, lactose high

38
Q

where can lac repressor bind?

A

at multiple operator sites(3)

O1 and O2 (these play major role in repression)

39
Q

lac repressor binding

A

tetramere
forms DNA-protein interaction
loops DNA out to prevent RNA pol from binding

40
Q

when allolactose bound to repressor

A

HUGE conf change

this dramatically decreases DNA binding affinity

41
Q

when glucose is low and lactose is high

A

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
Q

active activator helps…

A

RNA pol bind and transcribe genes needed to metabolize lactose

43
Q

what does cAMP do?

A

binds to CRP
turns activator on
(txn on)

binding to CRP bends the DNA

44
Q

what is adenylate cyclase activated by? What does it do?

A

low glucose
it makes cAMP

its a bacterial thing with a glucose binding domain

45
Q

Slide 11 for review

A

Slide 11 for review

46
Q

explain why adding glucose to a bacterial culture containing lactose inhibits beta-galactose expression

A

because transcription is off if glucose levels are high

47
Q

what happens of both glucose and lactose are low?

A

transcription is off

both activator and repressor are bound, but repressor wins

see experiment, almost new beta galactosidease is made

48
Q

what happens of glucose is low and lactose is high?

A

txn is on

49
Q

what happens if glucose and lactose are both high?

A

txn is off

50
Q

regulation of E. coli trp operon

A

1) transcriptional repression

2) transcriptional attenuation by ribosome stalling in mRNA

51
Q

how many amino acids needed to make tryptophan

A

5-6 depending on organism

52
Q

why would E. coli use TWO mechanism to regulate trp biosynthesis?

A

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
Q

trp operon

A

promoter
operator
leader sequence
5 genes that make the 5 enzymes

54
Q

trp repressor…

A

always being made
inactive in absence of tryptophan
works as repressor when there IS tryptophan

only make tryptophan if there is none

55
Q

where does trp repressor bind?

A

at operator site

56
Q

trp repressor only active repressor…

A

when bound to tryptophan!

when it is bound to tryp, it will bind to DNA and block transcription

57
Q

leader sequence

A

used with the attenuation mech
part of regulatory sequence
comes before the genes for txn
4 regions

58
Q

trp repressor

A

works just like the lac repressor pretty much

inactive when there is no tryp (so transcirption can happen)

59
Q 
transcriptional attenuation: 4 regions of leader sequence
region 1 (lots of tryptophan)
A

encoders for tryptophan

when ribosome gets here, if there is enough tryp around, RNA pol just keeps synthing

60
Q 
transcriptional attenuation: 4 regions of leader sequence
region 2 (lots of tryptophan)
A

if there is enough tryp around, the ribosome gets here quickly
Blocakge of sequence 2 before it can base pair with sequence 3

61
Q

transcriptional attenuation: 4 regions of leader sequence

region 3 and 4 (lots of tryptophan)

A

if ribosome is going fast, then 3 and 4 have to base pair,

hairpin structure between them forms

62
Q

what does formation of 3-4 hairpin structure do?

A

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
Q

transcriptional attenuation: when tryptophan levels are low

A

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
Q

2-3 base pair

A

DOES NOT act as attenuator

65
Q

Regulation of Bacillus trp operon

A

DOES NOT USE trp repressor protein or ribosome stalling

uses large RNA binding protein

repeated sequences in region upstream of genes

66
Q

what does Bacillus use of large RNA binding protein accomplish?

A

binds to tryptophan to halt transcription when trp levels are high

67
Q

Bacillus if lots of trp

A

tryptophan binding to TRAP (repeated protein that looks weird, binds to repeating RNA structure)
formation of hairpin structure
txn stops

68
Q

Bacillus if little trp

A

when transcribed to mRNA, normal structure forms, pol can read through region, txn happens

69
Q

TRAP binding (bacillus)

A

induces RNA hairpin to terminate transcription

70
Q

what might explain the evolution of yet another trp regulatory mechanism?

A

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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