The E-site story: Fundamental aspects of protein-synthesis Knud H. Nierhaus Max-Planck-Institute...

The E-site story: Fundamental aspects of protein-synthesis

Knud H. Nierhaus Max-Planck-Institute für Molekulare Genetik

Berlin-Dahlem, Germany

Tucson, 14th September 2006

MPI MOLGEN, NH GROUP

The E-site story: New aspects of the protein-synthesis

1.Introduction: Three tRNA binding sitesFunctional elements of the ribosomal elongation The ribosomal working plane of protein synthesis

2. Role of the E site I: molecular recognition and the solution of the ribosomal decoding riddle

3. Role of the E site II:Maintenance of the reading frame

16S RNA

21 proteinsS1 - S21

33 proteinsL1 - L36*

23S RNA 5S RNA

L7 = N-acetylated L12L8 = (L7/L12) L10L26 = S20

4

*


[C

]tRN

Abo

u nd

per

70S

1 4P

he

302010

1.0

2.0

3.0

040

tRNA

molar ratio tRNA:70S

plus mRNA

minus mRNA

Rheinberger, Sternbach & Nierhaus, 1980, PNAS 78:5310-5314

Hausner et al., 1988,J. Biol. Chem. 263:13103

The ribosomal interface provides a plane area for protein synthesis


Fitting of X-ray crystal structure of tRNA


Elongation Cycle Movie!

PRE POST

30S

50S

Agrawal et al., 2000, J. Cell Biol. 150:447-459

The Ribosomal Elongation Cycle3. Elongation

Cycle

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MPI MOLGEN, NH GROUP Agrawal et al., 2000, J. Cell Biol. 150:447-459


The E-site story: New aspects of the protein-synthesis machinery




Type I I I Type I Type I I


Types of molecular recognition

Nierhaus, 1993,Molecular Microbiology 9: 661-669

A U G G U A A AC C U G A C C A A G

P AE

Leu

Lys

Ala

fMet


P AE

Leu

Lys

Ala

fMet

ThrExplanations: aa–tRNA EF-Tu

Thr Ala Pro Ser Ser

Asn Ile Pro Pro Ser Ser Ala

Asp Cys Gly His Leu Phe Tyr

Val Arg Arg Ile Lys Met Arg

Arg Gln Gln Glu Gly Gly Leu

GTPGTP

GTP GTP GTP GTP GTP GTP GTP

GTP GTP GTP GTP GTP

GTP GTPGTP GTP GTP GTP GTP GTPGTPGTP



GTP GTP GTP GTP GTP GTP

Thr Thr

Leu Leu Trp ValLeu Sel-Cys

Nierhaus 74 Fig.2 /12.8.98



E site free and affinity at A sitehigh

cognate(1 species)

near-cognate(2-4 species)

non-cognate(~50 species)

E site occupied and affinity at A sitelow

cognate

near-cognate

non-cognate

G°

cogn

ate

near

-cog

nate

non-

cogn

ate

cogn

ate

near

-cog

nate

non-

cogn

ate

G°,

free

ener

gy o

f A-s

it e bi

ndin

g

contribution bytRNA-mRNAinteraction(codon-anticodon)

contribution bytRNA-ribosomeand Tu-ribosomeinteractions(A-site)

Nierhaus, 1990,Biochemistry, 29:4997


U

A C

G

E site free and affinity at A sitehigh

cognate(1 species)

near-cognate(2-4 species)

non-cognate(~50 species)

E site occupied and affinity at A sitelow

cognate

near-cognate

non-cognate

G°

cogn

ate

near

-cog

nate

non-

cogn

ate

cogn

ate

near

-cog

nate

non-

cogn

ate

G°,

free

ener

gy o

f A-s

it e bi

ndin

g

contribution bytRNA-mRNAinteraction(codon-anticodon)

contribution bytRNA-ribosomeand Tu-ribosomeinteractions(A-site)

Nierhaus, 1990,Biochemistry, 29:4997


Geigenmüller and Nierhaus., 1990,EMBO J. 9:4527


6000

4000

2000

fractions

200

400

600

800

E P A

POST

E P A

Pi

AcPheAsp AcPhe2

dpm [ H] per fraction ( )

3

dpm [ C] per fraction ( )

14

•EF-Tu•GTP

•EF-Tu•GTP+

•EF-Tu•GTP

•EF-Tu•GTP+

dpm

[14

C]P

he p

er f

ract

ion

dpm [

3H] A

sp per f rac tion


6000

4000

2000

fractions

200

400

600

800

E P A

POST

E P A

Pi

AcPheAsp AcPhe2


3


14

•EF-Tu•GTP

•EF-Tu•GTP+

•EF-Tu•GTP

•EF-Tu•GTP+

dpm

[14

C]P

he p

er f

ract

ion

dpm [

3H] A

sp per f rac tion


6000

4000

2000

fractions

200

400

600

800

E P A

POST

E P A

Pi

AcPheAsp AcPhe2


3


14

•EF-Tu•GTP

•EF-Tu•GTP+

•EF-Tu•GTP

•EF-Tu•GTP+


dpm

[14

C]P

he p

er f

ract

ion

dpm [

3H] A

sp per f rac tion



P AE

Leu

Lys

Ala

fMet


P AE

Leu

Lys

Ala

fMet

ThrExplanations: aa–tRNA EF-Tu

Thr Ala Pro Ser Ser

Asn Ile Pro Pro Ser Ser Ala

Asp Cys Gly His Leu Phe Tyr

Val Arg Arg Ile Lys Met Arg

Arg Gln Gln Glu Gly Gly Leu

GTPGTP


GTP GTP GTP GTP GTP

GTP GTPGTP GTP GTP GTP GTP GTPGTPGTP



GTP GTP GTP GTP GTP GTP

Thr Thr

Leu Leu Trp ValLeu Sel-Cys

Nierhaus 74 Fig.2 /12.8.98



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Valle et al., 2002,EMBO J. 21:3557


Conclusions 1: Importance of the E site for the decoding

The tRNA at the E site induces a low A-site affinity. This prevents the interference of non-cognate aa-tRNAs with the selection process. This means that a ribosome selects only one out of ≤4 aa-tRNAS (cognate+mis-cognate) instead of 1 out of ≈40.A mis-incorporation of a non-cognate aa-tRNA would be deleterious for the function of the corresponding protein in contrast to the mis-incorporation of a near-cognate aa-tRNA.Consequence of the E site effect: Selection of a near-cognate aa-tRNA destroys the function of a protein only 1 in 400 mis-incorporations due to the buffering of the codon lexicon.



Ogle et al., 2001,Science 292:897


The E-site story: New aspects of the protein-synthesis machinery




The importance of frameshift maintenance

5’- UUCUUCUUCUUCUUCUUCUUCUUCUUCUUCUUCUUCU -3’ 21 22 23 24 25 26

PhePhePhePhePhePhe

26 27 28 29 30 31 32

SerSerSerSerSerSerSer+1 frameshift


The mRNA from termination factor RF2

CUUAGGGGGUAUCUUUGACUCUGAUUCAAAAAGGGAU 5’- -3’ 21 22 23 24 25 26

26 27 28 29 30 31 32

Tyr LeuSTOP

Asp+1 frameshift


CUUAGGGGGUAUCUUUGACUCUGAUUCAAAAAGGGAU 5’- -3’ SD

21 22 23 24 25 26

26 27 28 29 30 31 32

Tyr LeuSTOP

Asp+1 frameshift

The mRNA from termination factor RF2


AAACUGGUUCCUUAGGGGGUAUCUUUGACUCUGAUUCAAAAAGGGAU

E P A

5’- -3’

Tyr Leu

SD

Termination: 25 aa peptide

+1 Frameshifting: Complete RF2 synthesis

A U A G A G

21 22 23 24 25 26

26 27 28 29 30 31 32

mRNA of the termination factor RF2


Hypothesis concerning the extensive frameshifting of theRF2 mRNA: 1


E P A

5’- -3’ 3‘-U C C U C C A’

Tyr Leu

SD A U A G A G

16S rRNA

ACUGGUUCCUUAGGGGGUAUCUUUGACUCUGAUUCAAAAAGGGAU



E P A

5’- -3’

Leu

SD G A G

Tyr

A U A

3‘-U C C U C C A’

16S rRNA



5’-


E P A

-3’ SD

G A G

Leu

C U G

Asp

+1 frameshift

3‘-U C C U C C A’

16S rRNA


Models of mRNAS for testing frameshifting of the RF2-mRNA type

mRNA with SD

Oligo(Phe) Arg Met Lys Leu Val Leu Val Leu Arg Gly Tyr Leu Stop GG UUC (UUC)11 CGU AUG AAA CUG GUU CUU GUU CUU AGG GGG UAU CUU UGA CUC UGA UUC AAA AAG GGA U

SD Asp Ser Asp Ser Lys Arg Asp

mRNA without SD Arg Met Lys Leu Val Leu Val Leu Arg Gly Tyr Leu Stop

GG UUC (UUC)11 CGU AUG AAA CUG GUU CUU GUU CUU CGC GGC UAU CUU UGA CUC UGA UUC AAA AAG GGA U

Ac[3H]Pheincorporation Tyr-

[32P]tRNAbinding

[14C]Aspincorporation= frameshift


Models of mRNAS for testing frameshifting of the RF2-mRNA type

Márquez et al., 2004, Cell 118:45-55

(UUC)12°°°°°°°°°°°°°°CGCGGCUAUCUUUGACUCUGAUUCAAAAAGGGAU

E P A3’-UCCUCCA

GAG

LeuTyr

AUA

mRNA

-SD

0

0.5

1

Aspincorporated

0.12 0.04

0.67 0.06

Synt

hesi

zed

pept

ide

chai

n (A

c[3 H

]Ph

e in

corp

orat

ion

)

E site tRNA[32P]

Frameshift[14C] Asp

incorporated


0.88 0.07

0

0.5

1

tRNATyr

0 0.03

(UUC)12°°°°°°°°°°°°°°AGGGGGUAUCUUUGACUCUGAUUCAAAAAGGGAU

E P A

3’-UCCUCCA

16S rRNA

SD GAG

Leu

Tyr

AUA

+SD


(UUC)12°°°°°°°°°°°°°°CGCGGCUAUCUUUGACUCUGAUUCAAAAAGGGAU

E P A3’-UCCUCCA

GAG

LeuTyr

AUA

mRNA

-SD

(UUC)12°°°°°°°°°°°°°°AGGGGGUAUCUUUGACUCUGAUUCAAAAAGGGAU

E P A

16S rRNA

SD GAG

Leu

CUG

Asp

Tyr

AUA

+SD

0

0.5

1

0

0.5

1

tRNATyr Aspincorporated

0.88 0.07

0 0.03

0.12 0.04

0.67 0.06

Synt

hesi

zed

pept

ide

chai

n (A

c[3 H

]Ph

e in

corp

orat

ion

)

E site tRNA[32P]

Frameshift[14C] Asp

incorporated


3’-UCCUCCA


CUU GUU CUU AGG GGG UAU CUU UGA CUC 70 E P

SDCUU GUU CAG GGG GUA UAU CUU UGA CUC 0 E P

SDCUU AGG GGG UGU GUA UAU CUU UGA CUC 0 E P

no SDCUU GUU CUU CGC GGC UAU CUU UGA CUC 6 E P

mRNAsFrameshift ratio

Asp incorp./ act. Ribosome%

SD StopTyr Leu

Shifting SD sequence upstream away from frameshifting site


Release of E-tRNA

Cause or Consequence?

gggaaaacaaaacaaaac- UAC-CUU-UGAC-aaaacaaaacaaaac

Tyr Leu Stop

YL stop mRNA

Asp

UAC C UUU GAC

E P AE site free

Fraction number

0 5 10 15 20 25 30 35 400

5

10

15

20

25

0

5

10

15

20

25

N-Ac[3H]Leu

[14C]Asp

(14 pmol)

N-Ac[3H]Leu-[14C]Asp

(38 pmol)

[14C

]Asp

[pm

ol]A

c[3 H

]Ph

e [p

mol

]

UAC CUU UGA C

E P A

?

+ GTP

GDP

?


UAC CUU UGA CE P A

?

E site occupied

GTP+


? UAC CUU U GAC

E P A

0 5 10 15 20 25 30 35 400

2

4

6

8

10

12

0

2

4

6

8

10

12

Fraction number

N-Ac[3H]Leu[14C]Asp

[14C

]Asp

[pm

ol]A

c[3 H

]Ph

e [p

mol

]

Conclusions 2: Importance of the E site for maintenance of the reading frame

The tRNA at the E site has to be removed in order to allow highly efficient frameshifting. The short spacer to the SD interaction blocks the –1 frameshift and probably fosters the +1 direction.Consequence: A tRNA at the E site and probably codon-anticodon interaction at the E site prevents mRNA slippage thus maintaining the reading frame.

Max-Planck-Institut für Molekulare Genetik,Berlin, Germany

Peter WurmbachHans-Jörg RheinbergerUte GeigenmüllerViter MarquezDaniel WilsonNils Burkhardt

Wadsworth Center, NY State Dept. of Health Albany, New York, USA

Joachim FrankRajendra AgrawalChristian SpahnMikel Valle

Pgggaaaacaaaacaaaac- UAC-UUC-UGAC-aaaacaaaacaaaac

Tyr Phe Stop

YF stop mRNAAsp

UAC UUC UGA C

E P A+ GTP UAC UUC U GAC

E P A

GDP

Fraction number

0 5 10 15 20 25 30 35 400

2

4

6

8

10

12

14

16

18

0

2

4

6

8

10

12

14

16

18N-Ac[3H]Phe

[14C]Asp

N-Ac[3H]Phe-[14C]Asp

E site free

Ac[

3 H]P

he

[pm

ol] [

14C]A

sp [p

mol]

ribosmal e

longation cycle

E P Aε δα

E P Aε α/δ

E P A

Tu•GTP

ε δα

POST

POST

PRE

PRE

E P Aε δα

E P Aε α/δ

TRANSLOC

ATION

TRAN

SFER

DECODING

•G GTP

• G GDP + Pi

•Tu GDP+Pi

Tu•GTPPOST

PRE POST

PRE

Dabrowski et al., 1998,J. Biol. Chem. 273:32793

RibosomalElongationCycle


The E-site story: Fundamental aspects of protein-synthesis Knud H. Nierhaus Max-Planck-Institute...

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