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