central dogma + regulation

1

central dogma

tRNA

rRNA

sRNA

mRNA

ribosomal proteins

DNApRNAp

structural proteins

transporters

regulators

enzymesDNA

rpl

tsx tsl

ribosomes

+ regulation

• tsx initiation control by transcription factors (TF)• tsl initiation control by sRNA and RNA-binding proteins • tsx termination control by anti-terminators (eg. protein, sRNA)• control of mRNA and protein degradation

coupled toenvironmentalsignals

1

tsx initiation controlby σ-factors and TFs

tsx elongation

tsx termination controlby proteins and sRNA

tsl initiation controlby proteins and sRNA tsl elongation

mRNA stability controlmRNA stability control

post-tsl control: modification & proteolysis

genetic circuits utilize allthese modes of regulation!

2

2

Topic 3: Post-transcriptional controlA. Transcriptional elongation and termination

1. Basic models of tsx elongation and termination2. mechanisms of termination (intrinsic vs rho-dependent)

B. Control of termination (=anti-termination or AT)1. AT at a single termination site (various mechanisms)2. processive AT (Q, N, Nus)

C. Translational mechanisms (initiation, elongation, termination)D. Translational control

1. RNA-binding protein2. riboswitch3. small regulatory RNA

E. Protein degradation and post-translational control1. proteolytic machinery2. protein unfolding3. substrate selection

3

A. Transcriptional elongation and termination(post-tsx-initiation control)

• “normal” termination at end of an operon• premature termination within or even at the beginning of an operon→ control mechanism (antitermination)

4

3

1. Model of tsx elongation

transcript length NN N+1N-1

backtracked

forward-trackedtra

nscr

iptio

n m

ode m

1

2

0

-1

energetics of tsx elongationΔGN ,m = ΔGN ,m; DNA bubble

+ΔGN ,m; RNA-DNA hybrid + ΔGN ,m; RNAP binding

! bubble prefers A/T-richstretches of DNA(AT: weaker basepair, DNA:DNA stronger than DNA:RNA hybrid)

! forwardtrack: energetically costly! backtrack: energetically neutral

(even favored if tsx error occurs)

5

kinetics of tsx elongation:• elemental pause -- freq: 1 every 10s-- duration: ~1s

• longer pauses-- freq: 1 every 100s-- duration: 1~10s

Adelman et al. 2002

Landick 2006

typ. few-tens sec.

in vitro also arrest

• pauses can be stabilized (and prolonged) by several mechanisms:

6

4

kinetic model:

N N+1

actively transcribing

paused

terminated

kfv

kfvT

⋅+

≈

⋅++

=

ττ00 1

1

)11(1

1

k

f 1/t

v0

Yager & von Hippel 1992

N-1

Termination efficiency

‘normal’ site: v0~100/s, f~0.1/s, t ~1s, k<0.1/s

termination site: v0~f~100/s, t~(1-10) s, k~ 1-5/s

T < 10-4

T~ 0.2-1

7

optimal terminator: • hairpin with GC-rich stem

followed by runs of U’s• 7-9nt separating hairpin and U’s• downstream sequence inductive

to TEC pausing

2. Mechanisms of termination(a) intrinsic termination

E. coli: 50% of mRNA have intrinsic terminators at their end70% of non-coding RNA mutations

disrupting termination

8

5

Model of intrinsic termination

• destabilization of TEC requires simultaneous disruption of HBS and RBS– hairpin formation displaces RNA from UBS (stimulated by NusA, part of TEC)– stretch of U’s provides weakest HBS

• pausing at terminator promotes hairpin formation and is essential for termination (depends on downstream sequence)

[Nudler & Gottesman, 2002]

9

(b) rho-dependent termination

• rho binds to ~40nt stretch ofunstructured, C-rich mRNA ( = RUT)

• translocation of rho requires energy• actual termination site not well-defined

– up to 120nt distal to RUT– generally correlate with pause sites

10

6

• polarity: nonsense mutation affects the expression of downstream genes

• tsx-tsl coupling → no tsx of untranslated mRNA(quality check on translation?)

• similar effect may arise at on-set of starvation (a.a. shortage)or upon exposure to translation-inhibiting antibiotics?

11

• polarity: nonsense mutation affects the expression of downstream genes

• tsx-tsl coupling → no tsx of untranslated mRNA(quality check on translation?)

• similar effect may arise at on-set of starvation (a.a. shortage)or upon exposure to translation-inhibiting antibiotics?

antibiotics or sudden starvation

RNAP

stalled ribosome

RNAP

rho-dependent termination

12

7

Quantitative study of tsx processivityDai, Zhu, TH (Nat Microb 2019)

LacZP O

LacIT0:IPTG

LacZP O RNAp

T1

LacZP O RNAp RNApT2

A B C

A AA

A

timeT1 T2T0

mR

NA

abun

danc

e

BB

B

C

13

0 40 80 120 1600

200

400

600

800

Induction time (s)

Lac

Z m

RN

A a

bu

nd

ance

P2365 primer

P259 primer

P1540 primer

P860 primer

P1903 primer

P3069 primer

P1199 primer

Glucose (DT: 45 min)

0 50 100 150 2000

200

400

600

800

Induction time (s)

Lac

Z m

RN

A a

bu

nd

ance

P1903 primer

P3069 primer

P1199 primer

P2365 primer

P259 primer

P1540 primer

P860 primer

Glucose FA 0.25 µg/mL (DT:200 min)

0 60 120 1800

200

400

600

800

1000

Induction time (s)

Rel

ativ

e m

RN

A le

vel

P1903 primer

P3069 primerP2365 primer

P259 primer


Glucose FA 0.1µg/mL (DT:100 min)

P1199 primer

0 60 120 1800

200

400

600

800

1000

Induction time (s)

Rel

ativ

e m

RN

A le

vel

Glucose 8µM Cm (DT:140 min)

P1903 primer


P259 primer

P1540 primer


A B

C D

waiting time (s)

Quantitative study of tsx processivity

x-intercept: (~10s)tsx init time

slope: tsx speed(47.6 nt/s)

position-dependentwaiting time

slope: d[m]/t

Dai, Zhu, TH (Nat Microb 2019)

14

8

0 40 80 120 1600

200

400

600

800

Induction time (s)

Lac

Z m

RN

A a

bu

nd

ance

P2365 primer

P259 primer

P1540 primer

P860 primer

P1903 primer

P3069 primer

P1199 primer


0 50 100 150 2000

200

400

600

800

Induction time (s)

Lac

Z m

RN

A a

bu

nd

ance

P1903 primer

P3069 primer

P1199 primer

P2365 primer

P259 primer

P1540 primer

P860 primer


0 60 120 1800

200

400

600

800

1000

Induction time (s)

Rel

ativ

e m

RN

A le

vel

P1903 primer


P259 primer



P1199 primer

0 60 120 1800

200

400

600

800

1000

Induction time (s)

Rel

ativ

e m

RN

A le

vel


P1903 primer


P259 primer

P1540 primer


A B

C D


0 40 80 120 1600

200

400

600

800

Induction time (s)

Lac

Z m

RN

A a

bu

nd

ance

P2365 primer

P259 primer

P1540 primer

P860 primer

P1903 primer

P3069 primer

P1199 primer


0 50 100 150 2000

200

400

600

800

Induction time (s)

Lac

Z m

RN

A a

bu

nd

ance

P1903 primer

P3069 primer

P1199 primer

P2365 primer

P259 primer

P1540 primer

P860 primer


0 60 120 1800

200

400

600

800

1000

Induction time (s)

Rel

ativ

e m

RN

A le

vel

P1903 primer


P259 primer



P1199 primer

0 60 120 1800

200

400

600

800

1000

Induction time (s)

Rel

ativ

e m

RN

A le

vel


P1903 primer


P259 primer

P1540 primer


A B

C D

0 40 80 120 1600

200

400

600

800

Induction time (s)

Lac

Z m

RN

A a

bu

nd

ance

P2365 primer

P259 primer

P1540 primer

P860 primer

P1903 primer

P3069 primer

P1199 primer


0 50 100 150 2000

200

400

600

800

Induction time (s)

Lac

Z m

RN

A a

bu

nd

ance

P1903 primer

P3069 primer

P1199 primer

P2365 primer

P259 primer

P1540 primer

P860 primer


0 60 120 1800

200

400

600

800

1000

Induction time (s)

Rel

ativ

e m

RN

A le

vel

P1903 primer


P259 primer



P1199 primer

0 60 120 1800

200

400

600

800

1000

Induction time (s)

Rel

ativ

e m

RN

A le

vel


P1903 primer


P259 primer

P1540 primer


A B

C D

0 50 100 150 2000

500

1000

Time (s)

Rel

ativ

e m

RN

A le

vel

Glucose Erythromycin 90 (µg/mL) (DT:126 min)

P1903 primerP3069 primerP1199 primerP2365 primerP259 primerP1540 primerP860 primer


15


0 1000 2000 3000 4000 50000.1250.2500.500

1248

16

Probe location

Slo

pe

Glucose FA 0.1FA 0.25Cm8C-Starvation

Z3' Y5' Y3' A5' A3'

• similar effects for different drugs and starvation• gradual loss of tsx processivity: 50% loss per 1-2 kb• abrupt drop (~4x) at the end of lacZ gene


16

9


0 60 120 1800

200

400

600

800

1000

Induction time (s)

Rel

ativ

e m

RN

A le

vel

Glucose 8 µM Cm

P1903 primer


P259 primer

P1540 primer


0 50 100 150 2000

200

400

600

800

1000

1200

Induction time (s)

Rel

ativ

e m

RN

A le

vel

Nonsense mutation

0 50 100 150 2000

200

400

600

800

1000

Induction time (s)

Rel

ativ

e m

RN

A le

vel

Glucose Ery 90 (µg/mL)

0 800 1600 2400 32000.0

0.2

0.4

0.6

0.8

1.0

Location

Rel

ativ

e sl

op

e

No drugFA 0.3FA 1.2Cm8

Isoleucine starvationNonsense mutation

Ery90

A B

D

C

0 100 200 3000

50

100

150

200

250

Induction time(s)

Rel

ativ

e m

RN

A le

vel

Isoleucine starvation

E

• similar effects for nonsense mutation (no physiological perturbation)• 50% loss per 0.5 kb

0 500 1000 1500 2000 25000.1

0.2

0.4

0.8

1.6

glucose Cm 8stop codon

position

mR

NA

synt

hers

is ra

te

LacZP O

nonsense mutation Dai, Zhu, TH (Nat Microb 2019)

17

0 50 100 1500

200

400

600

800

1000

Induction time(s)

LacZ

mR

NA

abu

ndan

ce

Stop codon-croRP297 primerP636 primerP898+150 primerP1237+150 primerP1578+150 primerP1941�150 primerP2403+150 primer

0 500 1000 1500 2000 25000

5

10

15

Position (nt)

Slo

pe

Stop codon croR terminatorA B


LacZP O

nonsense mutationtsx pause site (croR)

! effective termination by rho requires tsx pause site following stop codon! intended function of rho: termination vs quality control

0 50 100 1500

200

400

600

800

1000

Induction time(s)

LacZ

mR

NA

abu

ndan

ce

Stop codon-croRP297 primerP636 primerP898+150 primerP1237+150 primerP1578+150 primerP1941�150 primerP2403+150 primer

0 500 1000 1500 2000 25000

5

10

15

Position (nt)

Slo

pe

Stop codon croR terminatorA B


18

10

B. Control of termination: Anti-termination

• ~10% of genes in E. coli are controlled by AT• two types:-- control of a single termination site-- processive AT (controls many terminators)

• many different mechanisms

19

1. AT at a single terminator

positive feedback: increasesthe amplitude of fold-changes

BglG

alternative hairpin stabilizedby RNA-binding protein BglG

usually involves intrinsic terminator

(a) via protein-mRNA interaction (e.g., bgl operon)

alternative structure

20

11

(b) via sRNA-mRNA interaction

time window for sRNA action

anti-terminator

anti anti-terminator

[Gerhard Wagner lab]

with RNAIII

w/o RNAIII

21

(c) via small molecule-RNA interaction (riboswitch)

22

12

(d) via coupling to translation (= translational attenuation)

e.g., trp operon of E.coli (biosynthesis of Trp)

Trp level high: ribosome fast → termination

Trp level low: ribosome stalls → antitermination

23

translational attenuation

24

13

very different implementation of the same ‘idea’ in B. subtilis

trp RNA-binding attenuation proteinbound TRAP stabilizes terminator conformationno TRAP bound: AT more stable

25

2. Processive antitermination• requires special proteins (AT complex) which associate and travel with RNAP

• loading of ATC upstream of terminator• can read through multiple terminatorsover many thousands of bases

• well-studied examples:-- N and Q from phage lambda-- rRNA operons

N• N recruited by Nut site in RNA

Q

• Q recruited by qut site in DNA

• mechanism: stabilizes paused TEC

! Q and N necessary for transcribing long operons (Q: 23,000nt)

27

14

Q and N control long operons (Q: 23 kb !!!)

→ probability of spontaneous termination ?

estimate TEC stability using the model of Yager & von Hippel

• termination efficiency at normal site ~ 5 x 10-4

(possibly higher in vivo due to rho)

• normal operon (length < 5000 nt): 80 % of initiated transcripts reach end (if no internal termination sites)

• lambda late operon (23000 nt): 32 %

Q: 10-fold stabilization: restored to 90%

→ AT: control of operon + adaptation of TEC to operon length

28

Documents

central dogma + regulation