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3382 > Prokaryotic Regulation of Transcription > Flashcards

Prokaryotic Regulation of Transcription Flashcards

1
Q

bacteria can respond quickly to environmental changes because

A

transcription or repression of genes

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2
Q

strand of DNA that is read by RNAP is called

A

template strand

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3
Q

The strand that is not used to transcribe is called

A

the coding/non-template strand (identical to transcript except T-> U)

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4
Q

What are the units of the core enzyme of bacterial RNAP?

A

alpha2, beta, beta prime, omega

can function without omega

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5
Q

What is found in the active site of RNAP?

A

Mg2+, wich interacts with negatively charged bb

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6
Q

Steps of Transcription (bacteria)?

A
  1. binding
  2. initiation
  3. elongation
  4. termination
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7
Q

How does RNAP bind to DNA?

A
  • sigma subunit (holoenzyme) recognizes promoter sequences
  • RNAP binding protects nucleotide sequence from -70 to +20 where +1 is TSS. but binds to -10 and -35 region
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8
Q

is the transcription and translation start site the same?

A

NO

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9
Q

How does the Kd of the holoenzyme binding to DNA compre when DNA is wound vs unwound?

A

closed promoter complex (wound): 10^-9 M
open promoter complex (unwound): 10^-14 M

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10
Q

What are bacterial promoters?

A

defined nucleotide sequences that determine the site of transcription initiation (~40 nts on 5’ end)

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11
Q

What are the 2 consensus elements in bacterial promoters?

A

Pribnow bow (-10 region -> AT rich)
-35 region (variable – more AT rich = more efficient transcription = prob more necessary gene)

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12
Q

Is the sequence between -10 and -35 region important?

A

no – number of bases between are important otherwise RNAP would cover other important regions

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13
Q

Why are there different sigma factors?

A

each one recognizes a different -35 region

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14
Q

What are the steps of bacterial transcription initiation and elongation?

A
  1. sigma factor of RNAP recognizes DNA promoter
  2. formation of RNAP:closed promoter complex
  3. DNA begins to unwind at promoter -> open complex formation
  4. RNAP adds two NTPs (at initiation site and elongation site)
  5. 3’ OH of first NTP attacks a-phosphate of second forming phosphodiester bond
  6. elongation begins and sigma subunit dissociates
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15
Q

What are the two types of termination of bacterial transcription?

A
  1. Rho-independent/intrinsic termination
  2. Rho-dependent
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16
Q

How does Rho-independent/intrinsic termination work?

A
  • inverted repeat sequence rich in G-C bases form stable stem loop structure in RNA transcript
  • then a stretch of As in the DNA template (Us in the RNA) destabilizes RNAP bindind

termination sites present in DNA

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17
Q

How does Rho-dependent termination work?

what is Rho?

A
  • Rho translocates along RNA transcript until it reachs the transcription bubble
  • RNAP stalls in a G:C rich region of template DNA
  • Rho catches up and cause termination by allosterically changing RNAP and unwinding RNA from template DNA like a helicase

ATP-dependent helicase

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18
Q

Where do repressors and activators bind in bacteria?

A

repressors - operators (overlaps with promoter)
activator - enhancer (upstream of promoter)

repressors bind to silencers in eukaryotes

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19
Q

How does negative of gene expression work when induced or repressed?

A

induced: repressor always binds until an inducer inactivates repressor
repressed: repressor always binds and a corepressor promotes repression

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20
Q

How does positive of gene expression work when induced or repressed?

A

induced: activator always binds and an inducer promotes induction
repressed: activator always binds until a corepressor inactivates

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21
Q

What is the lac operon?

A

several genes under same promoter
translates into B-galactosidase transacetylase:
- transfer acetyl group from acetyl-CoA to B-galactosides

22
Q

What are the genes in the lac operon? What do they do?

A

LacY: translates to Galactosidate permease: channel for lactose to enter cell
LacZ: B-galactosization: hydrolyzes lactose to galactose or isomerizes to allolactose
LacI: represser

23
Q

What is glucose repression or catabolite repression?

A

preventing wasteful duplication of energy-producing enzymes by repressing transcription of lac and other operons

24
Q

What is the relationship between glucose and cAMP in glucose metabolism?

A

high glucose = low cAMP
and vice versa

cAMP increases when glucose runs out in a media

25
Q

the lac operon is under – regulation?

A

negative (repressed unless lactose is present and glucose is absent)

26
Q

Where exactly does LacI bind?

A
  1. strong operator site: O1, upstream of lacZ
  2. weak operator site: O3 upstream
  3. weak operator site: O2 downstream

there are 3 operator site for lac operon O1, O2, O3

27
Q

How does LacI binding affect RNAP?

A

LacI is a tetramer that can bind to 2 operator sequences at once

bind to 2 operators creates a loop in the DNA

loop prevents RNAP binding

28
Q

How is lac operon induced?

A
  • allolactose induces LacI so it can’t bind (changes its affinity for DNA)
29
Q

lacI mRNA is extremely…

A

unstable (1/2 life of 3 minutes, allows induction to be rapidly reversed to basal state)

30
Q

What are the 3 functional regions of LacI?

A
  • recognize and bind operator sequence
  • bind inducer at a site different from DNA binding site
  • oligomerization region that forms its 4º repressor structure
31
Q

since the Lac promoter is weak (weak interaction of sigma factor to -35 region) how is latose metabolism better induced?

A

catabolite activator protein (CAP) increases recruitment of RNAP
- CAP bound to CAP site on promoter
- RNAP’s a-subunit interacts with CAP to stabilize RNAP’s interaction with the promoter

32
Q

What is CAP regulated by?

A

cAMP!

adenylate cyclase is inhibited when glucose enters cell

when no glucose adenylate cylcase makes cAMP

cAMP binds to CAP which binds to enhancer sequence and to RNAP

33
Q

How is the arabinose operon (araBAD) regulated? When is it on and off?

A

genes/proteins: AraA, B, D
AraC = repressor
- no arabinose = OFF
- arabinose and glucose = OFF
- arabinose only = ON

34
Q

Where on the operon does AraC bind?

A

O2, O1, I2, I1 site throughout araBAD operon

35
Q

What does the araBAD operon look like?

A
  • has a promoter that works in 2 directions, one for araC and one for araB, A, D
  • O2, O1, I2, I1 sites for araC repression
36
Q

In the absence of L-arabinose, the AraC protein forms a

A

dimer that is stable when bound to I1 and O2 operator sites

37
Q

why is AraC dimer stable when bound to I1 and O2 operators?

when is it unstable?

A

forms a loop structure of ~210 bp between in the DNA that prevents RNAP binding

adding 5 bp to 210 = DNA in a half turn = binding site faces other way

38
Q

What happens to AraC when L-arabinose is present? How does it affect RNAP?

A
  • arabinose binds to AraC causing conformational change
  • this does NOT change protein-DNA affinity of AraC
  • this now makes AraC more stable when bound to I2 and I1 operators sites which are next to each other
  • this allows RNAP to bind but has to compete with AraC
39
Q

What is the activator of the araBAD operon? Why is it needed?

A
  • weak promoter
  • requires CAP-cAMP to activate when glucose levels are low
40
Q

How is AraC an autoregulator (along with a repressor) of its own transcription?

A

if a high level of AraC protein bind to O1 site, RNAP can’t RNAP is prevented from accessing the promoter for araC

41
Q

What is attenuation?

A

intrinsic terminators of transcription

42
Q

What does the trp operon look like?

A
  • attenuator sequence
  • each gene in the operon has its own stop codon before the next gene
  • each gene has a RBS at its start
  • each gene builds a small molecule in the trp making pathway

we need stop codons & RBSs otherwise erroneously make one long gene

43
Q

How is the trp operon regulated?

A
  • TrpR repressor
  • Tryptophan is a co-repressor
  • Tryptophan binds to TrpR which binds to trp operator

Transcription is not completely off!

44
Q

How is trp operon different from arabinose and lac operons?

A

default is ON
because loss of regulation = Trp is still being made in excess

45
Q

What is transcribed by trp operon when high Tryptophan levels?

A

makes a leader (TrpL) 139-nt segment

46
Q

What does the sequence of TrpL tell us?

A
  • encodes for 2 Trps in a row (Trp is rare, 2 in a row is very unusual)
  • started looking at other other amino acid synthesis operons and saw a similar pattern of multiple of its own AA in a row
47
Q

What structures do the trpL transcript make?

A
  • “terminator” (1:2 pause structure; 3:4 terminator)
  • “antiterminator” (2:3) - no terminator

2 different secondary structures from 4 regions

PAUSE STRUCTURE has Trp Trp and stop codon for leader peptide

48
Q

What does the terminator secondary structure of trpL look like?

A

an intrinsic terminator!
GC content stem loop structure and a poly(U) follows

49
Q

What happens in low tryptophan conditions?

A
  • ribosome binds to RBS and starts translating trpL
  • ribosome stalls when it gets to 2 successive trp codons in trpL -> incomplete leader peptide
  • ribosome sits ontop of sequence 1 (of 4)
  • ribosome pause = occlude seq 1
  • antiterminator forms where 2:3 seq makes hairpin loop
  • RNA polymerase continutes transcribing to make all trp genes which will make more tryptophan
50
Q

What happens in high tryptophan conditions?

A
  • ribosome translates trpL gene making a full leader peptide
  • no ribosome pause = occludes seq 1&2
  • 3:4 terminator bp structure forms
  • short transcript of just trpL gets made
  • transcription is teminated
51
Q

does the pause structure actually form?

A

no because the ribosome pauses on seq 2 – its just hypothetically what would form in ribosome wasn’t there

52
Q

which structure, antiterminator or terminator forms preferentially?

A

2:3 (antiterminator) has the chance to form first compared to 3:4 (in terminator)

also cell is default to ON to actually make trp

3382 (20 decks)

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