Рибосома как сенсор синтезируемого пептида и малых молекул

Рибосома как сенсор синтезируемого пептида
и малых молекул
Александр Манькин
Center for Pharmaceutical Biotechnology
University of Illinois at Chicago
The ribosome is an amazing RNA machine
Ada Yonath
Tom Steitz
Alexander Spirin
Harry Noller, Marat Yussupov,
Gulnara Yussupova
Venki Ramakrishnan
Elongation
Initiation
Termination
Schmeing)&)Ramakrishnan)(2009)))Nature,)461,)1234))
pep)dyl0transferase''
center'
nascent'pep)de''
exit'tunnel'
Nissen)et)al.,)(2000))Science,)289,)920;)Voorhees)et)al.,)(2009))Nat.Struct.Mol.Biol.,)16,)528;))Voss et al., (2006) J Mol Biol, 360, 893
Nissen)et)al.,)2000)Science)289,920)
Nakatogawa)&)Ito,)(2002))Cell,)108,629;)Gong)&)Yanofsky,)(2002))Science,)297,)1864;))Chiba)et)al.,)(2009))EMBO)J.)28,)3461;)Onouchi)et)al.(2005))Genes)Dev.)15,)1799))
Ribosome stalling controls expression of important bacterial and eukaryotic genes
Regulatory ORF
Regulated ORF
cofactor
cellular signal
secM
mifM
XBP1u
tnaC
CGS1
AAP
ermC
Nakatogawa)&)Ito,)(2002))Cell,)108,629;)Gong)&)Yanofsky,)(2002))Science,)297,)1864;))Chiba)et)al.,)(2009))EMBO)J.)28,)3461;)Onouchi)et)al.(2005))Genes)Dev.)15,)1799))
Nissen)et)al.,)(2000))Science,)289,)920;)Voorhees)et)al.,)(2009))Nat.Struct.Mol.Biol.,)16,)528;))Voss et al., (2006) J Mol Biol, 360, 893
Erythromycin (ERY)
Ketolides:Telithromycin (TEL)
O
N
N
N
O
O
OH
HO
N
OH
O
O
O
O
O
O
O
O
O
O
O
O
HO
N
OH
O
N
O
O
Schlunzen,)(2001))Nature,)413,814;)Tu)et)al.,)(2005))Cell,)121,)257;))Bukley)et)al.,)(2010).)PNAS)107,)17158;)Dunkle)et)al.(2010))PNAS)107,)17152))
Erm methyltransferase
A2058
CH3
H3C
NH2
N
N
H
N
N
N
N
N
H
N
N
Translation attenuation controls the expression of erm genes
Leader ORF
Spacer
3
I'I'I'I'I'I'I'I'
ermCL
2
I'I'I'I'I'I'I'I'
1
Resistance Gene
4
ermC
Non-Induced
2
3
I'I'I'I'I'I'I'I'
I'I'I'I''
1
ermCL
ermCL
1
I'I'I'I'I'I'I'I'
2
4
ermC
3
4
ermC
Induced
Horinouchi)&)Weisblum)(1980))PNAS)77,)7079;)Gryczan)et)al.(1980))Nucl)Acids)Res)8,)6081)
ermCL
16-17 nt
Ery
C C U - +
P site
ermCL
M G I F S I F V I S T V H Y …
Vazquez\Laslop,)et)al.)(2008))Mol.)Cell)30,)190)
Specific sequence of the nascent peptide controls stalling
key sequence
'MGIFSIFVI''
Vazquez\Laslop,)et)al.)(2008))Mol.)Cell)30,)190;)Ramu)et)al.)(2011))Mol.)Cell)41,)321))
tunnel
A2062
the most flexible residue in the tunnel
The mutant ribosomes are functionally active, but do not ‘recognize’ the nascent peptide!
Vazquez\Laslop,)et)al.)(2008))Mol.)Cell)30,)190;)Vazquez\Laslop,)et)al.)(2010))EMBO)J.)29,)3108)
Voorhees)et)al.,)(2009))NSMB,)16,)528;)Scheming)et)al.)(2005))Nature,)438,)520;))
A2062 and A2503 are sensors of the stalling signal in the tunnel
A2503
ERY
A2062
peptide
Modified nucleotides in 23S rRNA
m2A2503
NH2
N
N
HO
NH2
N
N
N
RlmN
N
O
H
H
H
OH
N
HO
O
H
N
OH
H
H
OH
OH
H
H
Toh)et)al.)(2008))RNA,)14,)98))
Posttranscriptional modification of the rRNA sensor contributes to the recognition of
the stalling signal
ermCL
wt'
ermC
rlmN)0'
inducible'ermC'
Vazquez\Laslop,)et)al.)(2010))EMBO)J.)29,)3108)
A2058
Erm methyltransferase
A2058
CH3
H3C
NH2
N
N
H
N
N
N
N
N
H
N
N
Modification of A2058 in the tunnel affects translation of several proteins
and reduces cell fitness
PoxA
poxA
Asn118
Gupta)et)al.)(2013),)Nature)Communica_ons,)in)press)
Where do sensors send the signal?
What becomes broken in the stalled ribosome?
peptidyl transferase center
A2503
ERY
A2062
peptide
ermCL
M''G''I''F''S''I''F''V''I'S''T''V''H''Y''Q''P''N''K''
ERY'
Ribosomal'
sensors'
I"
V"
F"
I"
S"
F"
I"
G"
fM"
Pep)dyl'
puromycin'
transferase'
center'
???'
Stalled ribosome is unable to catalyze peptide bond formation
Vazquez\Laslop,)et)al.)(2008))Mol.)Cell)30,)190)
Peptide and antibiotic affect the peptidyl transferase A-site
Peptidyl transferase center is the target of the stalling signal
Ramu)et)al.)(2011))Mol.)Cell)41,)321)
A'site'
C2452'
A2503
A2062
ERY
U2504'
A2503
P'site'
A2451'
G2061'
A2062
peptide
The'A2062'and'A2503'sensor'in'the'exit'tunnel'may'relay'the'stalling'signal'to'the'A0site'of'the'
pep)dyl'transferase'center'
Translation attenuation control requires the antibiotic
Leader ORF
Spacer
3
I'I'I'I'I'I'I'I'
ermCL
2
I'I'I'I'I'I'I'I'
1
Resistance Gene
4
ermC
Non-Induced
2
3
I'I'I'I'I'I'I'I'
I'I'I'I''
1
ermCL
ermCL
1
I'I'I'I'I'I'I'I'
2
4
ermC
3
4
ermC
Induced
Horinouchi)&)Weisblum)(1980))PNAS)77,)7079;)Gryczan)et)al.(1980))Nucl)Acids)Res)8,)6081)
What is the role of the antibiotic in nascent peptide-dependent ribosome stalling?
2
3
ermCL
I'I'I'I'I'I'I'I'
I'I'I'I''
1
!!!
A2503
A2062
A road-block
4
ermC
ERYTHROMYCIN'
CLARYTHROMYCIN'
O
TELITHROMYCIN'
O
OH
HO
O
O
O
O
O
O
O
O
O
O
HO
OH
O
O
OH
O
O
O
O
O
O
O
O
O
O
O
HO
OH
O
O
O
O
O
HO
OH
O
O
O
O
O
HO
N
O
O
ITR163'
O
O
OH
O
ITR162'
N
N
O
ITR156'
O
N
HO
OH
ITR166'
O
O
O
O
O
O
O
HO
N
OH
O
HO
N
OH
O
O
O
N
O
O
N
OH
HN
O
N
OH
HN
O
N
OH
HN
O
N
O
O
Vázquez\Laslop)et)al.)(2011),)PNAS,)108,)10496'
OH
U1782
A2602
C2610
A2503
A2059
A2058
Specific tunnel sensors recognize the presence of the stalling cofactor and its structure
Vázquez\Laslop)et)al.)(2011),)PNAS,)108,)10496'
✕
Peptidyl
transferase
The analog signal from the peptide and small molecular cofactor is integrated in the
peptidyl transferase center
Nascent peptide defines the recognition of the small molecular cofactor
MGIFSIFVISTVHYQPNKK
19'
ermCL)
ermC'
MLVFQMRNVDKTSTI…
36'
ermB'
ermBL)
✔
✔
Erythromycin (ERY)
Telithromycin (TEL)
O
N
N
N
O
O
OH
HO
N
OH
O
O
O
O
O
O
O
O
O
O
O
O
HO
N
O
OH
N
O
✔
✗
O
ErmCL: M G I F S I F V I . . . . . . !
!
!
!
ErmBL: M L V F Q M R N V D . . . . .!
!
!
!
CL-BL: M G I F S M R N V D . . . . . !
!
'
ERY
TEL
✔
✗
✔
✔
✔
✔
The potential to discriminate between the cofactor structures resides in the
C-terminus of the stalling nascent peptide
Erythromycin Telithromycin
F
Y
N
G
Q
E
T
stalling
P P
WW
M L V F Q M R N V D
A
R
C
H
I
L
K
M
S
V
F
Y
A
R
C
H
I
L
K
M
S
V
N
G
Q
E
T
no stalling
ErmBL: M L V F Q M R N V D . . . . .!
Telithromycin
Stalling
peptide
R N V D!
Ery
Tel
O
N
N
N
+''
+'
R N V E!
+'
0'
R N V Y!
0'
+'
R N V A!
0'
0'
Erythromycin
O
O
OH
HO
OH
N
O
O
O
O
O
O
O
O
O
O
O
O
O
HO
N
O
N
OH
O
The nature of the P-site amino acid defines what small molecule can be
recognized as a stalling cofactor.
Small molecule can affect the discriminating properties of the ribosome
peptidyl-tRNA
drop-off
Macrolides stop protein synthesis at the early rounds of translation by
promoting drop-off of peptidyl-tRNAs with short nascent peptides
No'an)bio)c'
Erythromycin'
Telithromycin'
O
N
OH
HO
N
N
OH
O
O
N
O
O
O
O
O
O
O
O
O
O
HO
O
N
OH
O
O
O
N
O
Specific proteins can be synthesized y the drug-bound ribosome.
The structure of the antibiotic in the tunnel affects the spectrum of synthesized proteins
Kannan)et)al.)(2012),)Cell,)151,)508'
! Специальные нуклеотидные сеносры в туннеле позволяют рибосоме
«узнавать» определенные структуры в синтезируемом белке.
! Антибиотик помогает рибосоме распознать такие структуы, но и сам
антибиотик распознается как сигнал остановки трансляции.
! Меняя структуру белка, закодированого в мРНА можно научить
рибосому узнавать разные низкомолекулярнуе соединения.
! Таким образом рибосома выступает в качестве сенсора и вновь
синтесируемого пептида и некоторых малых молекул
shura@uic.edu
Nora Vazquez-Laslop
Mashal Almutairi
Krishna Kannan
Dorota Klepacki
Pulkit Gupta
Teresa Szal
Collaborations:
Tanel Tenson (Tartu University)
Ada Yonath (Weizmann Institute)
Jonathan Weissman (UCSF)
Priya Sothiselvam
Tom Steitz (Yale)
Daniel Wilson (U of Munich)
Klaus Schulten (U of Illinois)