RNAs Catalysing Chemical Reactions - Ribozymes · Reactions - Ribozymes. autocatalytic group I...
Transcript of RNAs Catalysing Chemical Reactions - Ribozymes · Reactions - Ribozymes. autocatalytic group I...
RNA Catalysis
RNAs Catalysing Chemical Reactions - Ribozymes
autocatalytic
group
I introns(Tetrahymena
Ribozyme)
RNase
P from
E. coli
1982: discovery
of the
catalytic
activity
of RNA by
Thomas Cech
and Sidney
Altman
RNA Ribosomale
(rRNA)Transfer (tRNA)Messenger (mRNA)
snRNAs (U1, U2, U4, U5, U6)sno
RNAs (~ 200)Ribozyme (HH, HDV, HP, VS)Gruppe
I und II IntronsRNase
PtmRNA, SRP-RNAXist RNA, roX RNAsregulatorische
antisense RNAs (>500)nicht
kodierende
RNA (>500)synthetisch
selektierte
RNAs (>100)
RNomics
RNA molecules
fold
into
complex
tertiary
structures
Ribozymes:
Large Ribozymes: group I intron, group II intron, spliceosome,RNAse
Pribosome,
Small ribozymes: hairpin, delta virus, hammerhead Ribozymes isolated via SELEX
RNA catalysis: natural and synthetic enzymes
Mechanism of RNA catalysis:
2 Models:
1. Metal ion catalysis
2. Acid base catalysis andbases with disturbed pKa
Fedor
and Willamson
NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 6 | MAY 2005 | 401
Concerted general acid–base catalysis by ribonuclease A Ribonuclease (RNase) A is a protein enzyme that catalyses the same chemical reaction as self-cleaving RNAs. RNase A provides a textbook example of concerted general acid–base catalysis90. Residue His12, in its unprotonated form,functions as a general base catalyst to remove a proton from the attacking 2.- oxygen nucleophile, whereas His119, in its protonated form, functions as a general acid catalyst to protonate the 5.-oxygen leaving group. As shown in the figure, a hydrogen-bonding interaction between the positively charged ε-amino group of Lys41 and the nonbridging phosphoryl oxygen provides electrostatic stabilization to the transition state (represented by ‡).
Acid-base catalysis in a protein enzyme
RNase A
Fedor
and Willamson
NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 6 | MAY 2005 | 401
Nucleotides and acid-base catalysis
acid-base
catalysis
pKa
Verschiebungenführen zu protoniertenBasen bei pH
7!
Metal ion catalysis
Fedor
and Willamson
NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 6 | MAY 2005 | 401
Divalent metal cations, such as Mg2+, can interact with RNAs through inner sphere (direct) coordination to phosphate and ribose oxygens. In this way, they can activate nucleophilic
oxygens
during oxygen–phosphorus bond formation or stabilize oxyanion
leaving groups during oxygen–phosphorus bond breakage, and counternegative charges that develop on nonbridging
oxygens
in transition states. Diffusely associated metal cations
also interact nonspecifically with RNAs and facilitate the assembly of functional structures by neutralizing the phosphate charge. Mg2+ is aparticularly effective counterion
for stabilizing RNA structures because of its smallsize and high charge density.
Metal ion catalysis
Metal ion hydroxide as nucleophile source
MeH2
0 MeOH- + H+
How can
the
interaction
of a metal ion
with
RNAbe
determined?
1)
X-ray
structure2)
phosphothioate
chemistry
Magnesium (hard metal) interacts well with oxygen (hard ligand
butpoorly with soft ligands
like sulfur and nitrogen.
Soft metal ions like Cadmium and Manganese interact well with softligands.
„Manganese
rescue“
Autocatalytic Goup I Introns
Catalytic RNAs
2-dimensional 3-dimensionalMg2+
Fedor
and Willamson
NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 6 | MAY 2005 | 401
Mechanism
of selfsplicing
Group II Intron andpre-mRNA splicing
Group II Intron Secondary
Structure
g
g
G
GG
G
G
G
GG
G
G
g
G
g
GG
g
a
A
A
AA
AA
AA
AA AA
a
a
a
uuu
u
u
U
UU
U
U
U
uU
U
CC
C
C
C
C
c
C
C
AAU
G
U
A
A
UAAU
AA
A
A
U
U
A
A
A
A
A
A
U
U
U
U
U
U
U
U
UUA
AU
UA
A
AA
AA
UUU
U
GA AAGA
AU
AG
AU A
AAG AUUA
AA
UAAUUU UA
AAUUA
U
UAUG
UU
UACAA
UAU A U
AU
ACCU
AAU
UA
G
A
UGGAA
U
U
AG
A
AAUCUCA
AACAUGCAUAUGU
U
UUAU U UAG
AAUAAUU
AAUAAUUAUUAUUAAUUA
UU
GAUUCAU U
AAGUG
AUA
GAU AA
UCCUAAU
AGCGU
AAGU
CAACUAAU
GAUUAAUUA
UAA AA
AU
UCG
AA
AU
UAA
AUGGCU
UUC
UA
AA CA
U
AA
UUU
UU
U
U
AA
U
ACUC
GAG
AA
AAAAA
UUU C
UAAAA AU UU UAUAA AU UA
A U UAAU
AA
AA G U A A G
A
UA UUC
AU
A
AUUGC AUAU
UC
AA
A
UAA G
AAUUC
C
UU
GUA
CCCAA
UUAU
UUCG
GGGUGAAUA
GAUG
UAA U
A
CAUU
AA
UAA
AUAUU
GUUUAUAAAU
AU AA
UUAUA
AAUA
UAUA
A
UUAA
GU AA
AU
UU
UA
A
U
UA
AA
AUA
AU AAA
UA
A UUA
UUGAA
AACUU
AAAGC
CAA
UCGUACG A A
AGUGUAU
AAAGAUUAAUU
GUAUAAU
GUAAU
A
AUA
U
GAUCAAA
A
A
A
AU
U
U
U
U
U
UU
U
UU
UU
UU
UUAGUAAAUAAGC
AAU U UA A A
A
A
A
AA A A
A
A
A
A
UUUAUUAUUAU
UU
AA
AGG
U
AUUUU A
AAAUAUAUU
AAUAA
AA U
A
AUGAU
UUAUA
UGUUAAAUAU
U AGU
A
AGCUGUA
UUGACAUG C
UA
AACUAU
U
UG
G AAAG
UGGGGGAA AAUUU
CUAUCC
UAUC AGA
A
* GU
AA
A
IBS2
IBS1
AEBS2
EBS1
α
α’
I
II
III
IV
V
VI
Cob bI1
5’ss 3’ss
ζ’
ζκ’
κ
ο’
ο
γ’
γ
β’
β
η'
η
δ'
δ
Spliceosome assembly
Patel
and Steitz, Nature reviews, Molcellbio, 4, 960
+ ~200 non-snRNPproteins
Similarities between domain 5 of group II self-splicingintrons
and the U6 ISL
UV crosslink
Hypothesis: group II introns and spliceosome have evolved from a common ancestor
●
identical reaction pathway
●
identical stereochemistry
●
same requirement for divalent metal ions (Mg 2+)
●
metal ion stabilizes the 3’-
oxyanion
leaving group
21
3
1 RNase
P (Endonuklease)2 Exonuklease3 CCA-
Addition tRNA nucleotidyltransferase
Processing of tRNAs
RNAse P
RNA components from RNase Ps
Hammerhead
HepatitisDelta VirusRibozym
HairinRibozym
NeurosporaVS Ribozym
Hammerhead Ribozyme
Hairpin ribozyme structure and mechanism.Secondary structure diagram of the two-way junction and the four-way(natural) junction form of the hairpin ribozyme, and the crystal structure of the hairpin ribozyme
complex with a noncleavable
substrate analogue15. Arrows highlight the reactive phosphodiester. The colours
highlight: G+1, A–1 and the reactive phosphodiester (yellow); domain A (blue); domain B (red); and the extra helices that form a four-way helical junction (purple). With respect to A–1, an adenosineis at this non-conserved (N) position in the natural hairpin ribozyme
and the RNA used for crystallization. The green spheres represent two bound calcium ions.
Peptidyltransfer
(1)
Peptidyltransfer
(2)
Active sites on 50S and 30S subunit
Ramakrishnan (2002) Cell 108; 557
Transitionstate
(TSA) Transition
state analogon
P. Nissen et al., Science 289, 11. Aug. 2000, Cover
PTC and proteins
Distance of PTC to proteins
Secondary
and tertiary
structures
of 23S and 5S RNA
Yusopov, M.M. et al., Science (2001) 292, 883
The peptidyl transferase site
Nissen, P. et al., Science (2000), 289, 920-930
How can RNA catalyse
peptide bond formation?
OHOO
N R
A-siteHOOO
NH R
OR
NHAc
P-site
- -+
-
H
H
B-H
:B
B-H
Protons (H+)Hydrated metals
HN
N N
N
NH2
HN
N
NH2
O
Cytosine Adenine
Histidine
HN NH
-OOCNH3
N
N
H
Imidazole
Hydroxide ions (OH–)Hydrated metals
N
N N
N
NH2
N
N
NH2
O
Cytosine Adenine
: :
Histidine
:N NH
-OOCNH3+
N
N
Imidazole
Models for catalysis of peptide bond formation
Model for metal ion catalysis of peptide bond formation
OHOO
N R
A-site
HOOO
NH R
OR
NHAc
P-site
Mg2+
Mg2+
Mg+
H
HO
H
δ–
δ–
δ–δ+
Mg(H2 0)x2+
OHOO
N R
A-site
OHSS
NH R
OR
NHAc
P-site
Mn2+
Mn2+H
H
Manganese rescue
Mononucleotide substrates for testingpeptidyl transferase activity
N
N
N
N
OH
O
NH2
OO
AcNH R
P
N
N
N
N
S OH
O
AcNH R
S
P
NH2
N
N
N
N
O OH
O
NH2
AcNH R
S
P
N
N
N
N
S
O
NH2
AcNH R
O
P
O
AcNH R
O
The significance of the 2‘-OH of P-site tRNA A76
Possible involvement of the 2´-OH in catalysis of peptide bond formation
Peptidyl transferase Center