CHAPTER 7 - CATALYSIS
E: RIBOZYMES and the RNA World
BIOCHEMISTRY - DR. JAKUBOWSKI
Learning Goals/Objectives for Chapter 7E: After class and this
reading, students will be able to

define a ribozyme and describe their known activities;

identify, given a reaction mechanism, the types of catalytic
mechanisms that occur during ribozyme catalysis;

contrast the chemical and physical properties of dsDNA, ssRNA, and
proteins and how they may confer on these polymers critical attributes
necessary for their biological functions/activities;

give reasons that would explain how simple life might have originated
using RNA, not DNA and proteins, as both the carrier of genetic
information and as biological catalyst.
"A foolish consistency is the hobgoblin of little minds...." Ralph Waldo
Emerson
Ribozymes
Any molecule that displays any of the catalytic motifs seen in the earlier chapters
(general acid/base catalysis, electrostatic catalysis, nucleophilic catalysis, intramolecular
catalysis, transition state stabilization) can be a catalyst. So far we have examined only
protein catalysts. These can fold to form unique 3D structures which can have active
sites with appropriate functional groups or nonprotein "cofactors" (metal ions, vitamin
derivatives) that participate in catalysis. There is nothing special about the ability of
proteins to do this. It is now known that RNA, which can form complicated secondary
and tertiary structures as seen in the 3D image of the ribozyme from Tetrahymena
thermophila, can as well.
Figure: ribozyme from Tetrahymena thermophila,
RNA molecules that act as enzymes are called ribozymes. This property of some RNA's
was discovered by Sidney Altman and Thomas Czech, who were awarded the Nobel
Prize in Chemistry in 1989. In contrast to protein enzymes which are true catalysts in
that they are used over again, this is an example of a single use ribozyme. Other
ribozymes are true catalysts and can carry out RNA slicing by transesterification
(splicesome) and peptidyl transfer (in ribosomes). The mechanisms of catalysis of the
hepatitis delta virus ribozyme include general acid/base catalysis.
Figure: mechanisms of catalysis
The hairpin ribozyme from satellite RNAs of plant viruses is 50 nucleotides long, and can
cleave itself internally, or , in a truncated form, can cleave other RNA strands in a
transesterification reaction. The structure consists of two domains, stem A required for
binding (self or other RNA molecules) and stem B, required for catalysis. Self-cleavage in
the hairpin ribozyme occurs in stem A between an A and G bases (which are splayed
apart) when the 2' OH on the A attacks the phosphorous in the phosphodiester bond
connecting A and G to form an pentavalent intermediate.
Figure: Self-cleavage in the hairpin ribozyme
Rupert & Ferré-D'Amaré (2001) solved the crystal structure of a hairpin
ribozyme with a non-cleavable substrate analog containing a in which a 2'OCH3 was substituted for the nucleophilic 2'-OH group.
See the applet
below.
A recent study by Rupert et al. (2002) shows that A38 in Stem B appears to be able to
interact with the products (the cleaved A now in the form of a cyclic phosphodiester with
itself) and the departing G, and also with a transition state pentavalent analog of the
sessile A-G bond in which the phosphodiester linking A and G in the substrate is
replaced with a pentavalent vanadate bridge between A and G. However, A 38 does not
appear to react with the sessile A -G groups in the normal substrate, indicating that the
main mechanism used by this ribozyme is transition state binding. Since RNA molecules
have fewer groups available for acid/base and electrostatic catalysis (compared to
protein enzymes), ribozymes, presumably the earliest type of biological catalyst, probably
make more use of transition state binding as their predominant mode of catalytic activity.
Figure: Active Site of Hairpin Ribozyme: Transition State Binding
Recently, the crystal structure of a purple bacterium group I self-splicing intron (which
catalyze the removal of itself) interacting with both exons in a state prior to their ligation
was determined (Adams, P. et al.). The structure shows both exons in close proximity.
Nucleophilic attack of the 3'OH of the 5' exon on a distorted phosphate at the intron-3'-
exon junction.
Two metal ions reside on either side of the labile phosphodiester bond at
the intron-3'exon junction, and are held in place by 6 phosphates.
A novel use of ribozymes was recently reported by Winkler et. al. They discovered that
the 3' end of the mRNA of the gene glmS (from Gram-positive bacteria) which
encodes an amidotransferase (catalyzing the formation of glucosamine-6-phosphate from
glutamine and fructose-6-phosphate) is a ribozyme. A glucosamine-6-phosphate
binding site in the ribozyme (3' end of the mRNA) binds this sugar, inducing autocleavage
of the ribozyme. This inhibits, by an uncertain mechanism, the formation of the
amidotransferase from the remaining part of the mRNA. This mechanism of regulation of
gene expression through ribozyme activity might prove to be common.
In order for RNA molecules to have acted as both catalysts and carriers of
genetic information in the original RNA world, RNA must have the potential to
self replicate. Shechner et al. looked at the core structure of a class I ligase
ribozyme able to polymerize RNA. This ligase was synthesized and enhanced
through
The tripod structure allows more RNA solvent interaction then often
found in ribozymes. Several common structural motifs were also found to be
present including the GNRA-triloop, uridine-turn and the frameshifting
pseudoknot motifs. GNRA-triloop and uridine-turn motifs are short
thermodynamically stable segments that cap the end of the helical regions.
Varieties of these are found in many RNA structures although they don’t seem
to be necessary for activity. The frameshifting pseudoknot motif is almost
identical in structure to small viral ribosomal frameshifting pseudoknots and is
adjacent to a new motif named “A-minor triad”. The A-minor triad is
responsible for the coordination with Mg2+, although only when inserted inbetween certain sequences. Triphosphoguanosine (called G1) at the 5’
terminus of the ribozyme acts as the electrophile for the RNA ligation
reaction. Other segments of the ribozyme, such as the J1/2, have highly
specific contacts which orient G1 and allow the RNA to fold more efficiently.
Two residues, cytosine 12 (C12) and uracil 48 (U48) bind to Mg2+. Other
sections of the RNA also promote specificity to assist in the replication
process. A model for catalysis and the transition state of the ribozyme
polymerase is similar to that for protein RNA polymerases, as shown in the
figure below
Figure: Comparison of Transition State Models of Ribozyme and Protein RNA
Polymerases (after Schechner et al)
A divalent Mg2+ in the active site of the ribozyme enhances the nuclophilicity
of the 3-OH on the primer, which attached the the terminal phosphate of the
G(1)TP substrate to form a pentavalent intermediate. The Mg cation is
stabilized by oxygens on P 29 and 30 of the ribozyme. The Mg ion also
stabilizes the developing charge in the transition state and in the charge in
the intermediate. Stabilization of analogous divalent cations in the protein
polymerase occurs through Asp side changes in the protein.

Hairpin Ribozyme Add 1HP6.pdb
Jmol: Self-splicing Group I intron with both exons
Jmol:
L1 Ligase Ribozyme

3D Structure of Ribozyme with Active Site - From the Howard
Hughes Medical Institute.
12/1/10 The Biggest Ribozyme of them all: The Ribosome
Protein synthesis from a mRNA template occurs on a ribosome, a
nanomachine composed of proteins and ribosomal RNAs (rRNA). The
ribosome is composed of two very large structural units. The smaller unit
(termed 30S and 40S in bacteria and eukaryotes, respectively) coordinates
the correct base pairing of the triplet codon on the mRNA with another small
adapter RNA, transfer or tRNA, that brings a covalently connected amino
acid to the site. Peptide bond formation occurs when another tRNA-amino
acid molecule binds to an adjacent codon on mRNA. The tRNA has a
cloverleaf tertiary structure with some intrastranded H-bonded secondary
structure. The last three nucleotides at the 3' end of the tRNA are CpCpA.
The amino acid is esterified to the terminal 3'OH of the terminal A by a
protein enzyme, aminoacyl-tRNA synthetase.
Covalent amide bond formation between the second amino acid to the first,
forming a dipeptide, occurs at the peptidyl transferase center, located on the
larger ribosomal subunit (50S and 60S in bacteria and eukaryotes,
respectively). The ribosome ratchets down the mRNA so the dipeptide-tRNA
is now at the the P or Peptide site, awaiting a new tRNA-amino acid at the
A or Amino site. The figure below shows a schematic of the ribosome with
bound mRNA on the 30S subunit and tRNAs covalently attached to amino acid
(or the growing peptide) at the A and P site, respectively.
Figure: Prokaryotic Ribosme - P and A sites
A likely mechanism (derived from crystal structures with bound substrates
and transition state analogs) for the formation of the amide bond between a
growing peptide on the P-site tRNA and the amino acid on the A-site tRNA is
shown below. Catalysis does not involve any of the ribosomal proteins (not
shown) since none is close enough to the peptidyl transferase center to
provide amino acids that could participate in general acid/base catalysis, for
example. Hence the rRNA must acts as the enzyme (i.e. it is a ribozyme).
Initially it was thought that a proximal adenosine with a perturbed pKa could,
at physiological pH, be protonated/deprotonated and hence act as a general
acid/base in the reaction. However, none was found. The most likely
mechanism to stabilize the oxyanion transition state at the electrophilic
carbon attack site is precisely located water, which is positioned at the
oxyanion hole by H-bonds to uracil 2584 on the rRNA. The cleavage
mechanism involves the concerted proton shuffle shown below. In this
mechanism, the substrate (Peptide-tRNA) assists its own cleavage in that the
2'OH is in position to initiate the protein shuttle mechanism.
(A similar
mechanism might occur to facilitate hydrolysis of the fully elongated protein
from the P-site tRNA.) Of course all of this requires perfect positioning of the
substrates and isn't that what enzymes do best? The main mechanisms for
catalysis of peptide bond formation by the ribosome (as a ribozyme) are
intramolecular catalysis and transition state stabilization by the appropriately
positioned water molecule.
Figure: Mechanism Peptide Bond Formation by the Ribosome
The crystal structure of the eukaryotic ribosome has recently been published
(Ben-Shem et al). It is significantly larger (40%) with mass of around 3x106
Daltons. The 40S subunit has one rRNA chain (18) and 33 associated
proteins, while the larger 60S subunit has 3 rRNA chains (25S, 5.8S and 5S)
and 46 associated proteins. The larger size of the eukaryotic ribosome
facilitates more interactions with cellular proteins and greater regulation of
cellular events. The Jmol structure of a bacterial 70S ribosome showing
mRNA and tRNA interactions is shown below.
Jmol: 70 S Ribosome from Thermus Thermophilus showing mRNA and tRNA
interactions
: Ribosome in Action
The RNA World
Given that RNA expresses catalytic activities and can carry genetic information (some
viruses have ds and ss RNA as their genome), it has been suggested that early life
might have been based on RNA. DNA would evolve later as a more secure carrier of
genetic information. An inspection of chemical properties of DNA, RNA, and proteins
shows them to have attributes needed for their expressed function. Let's examine each
for structural features that might be important for function.
a. Why does DNA lack a 2' OH group (found in RNA), which has been replaced with a
hydrogen? This required the evolutionary creation of a new enzyme, ribonucleotide
reductase, to catalyze the replacement of the OH in a ribonucleotide monomer to form
the deoxyribonucleotide form. One possible explanation if offered in the figure below.
DNA, the main carrier of genetic information, must be an extremely stable molecules. An
OH present on C'2 could act as a nucleophile and attack the proximal P in the
phosphodiester bond, leading to a nucleophilic substitution reaction and potential
cleavage of the link. RNA, an intermediary molecule, whose concentration (at least as
mRNA) should rise and fall based on the need for a potential transcript, should be more
labile to such hydrolysis.
b. Why do both DNA and RNA contain a phosphodiester link between adjacent
monomers instead of more "traditional" links such as carboxylic acid esters, amides, or
anhydrides? One possible explanation is given below. Nucleophilic attack on the sp3
hybridized P in a phosphodiester is much more difficult than for a more open sp2
hybridized carboxylic acid derivative. In addition, the negative charge on the O in the
phosphodiester link would decrease the likelihood of a nucleophilic attack. The negative
charges on both strands in ds-DNA probably helps keep the strands separated allowing
the traditional base pairing and double stranded helical structure observed.
c. Why is DNA found as a repetitive double-stranded helix but RNA is usually found as a
single stranded molecule which can form complicated tertiary structures with some dsRNA motifs?
Another reason for the absence of the 2' OH in DNA is that it allows the
deoxyribose ring in DNA to pucker in just the right way to sterically allow
extended ds-DNA helices (B type).
The pucker in deoxyribose and ribose can
be visualized by visualizing a single plane in the sugar ring defined by the ring
atoms C1', O and C4'.
If a ring atom is pointing in the same direction as the
C4'-C5' bond, the ring atom is defined as endo. If it is pointing in the
opposite direction, it is defined as exo (see Jmol below). In the most
common form of double-stranded DNA, B-DNA, which is the iconic extended
double helix you know so well, C2' is in the endo form. It can also adopt the
C3' endo form, leading to the formation of another less common helix, more
open ds-A helix.
In contrast, steric interference prevents ribose in RNA from
adopting the 2'endo conformation, and allows only the 3'endo form,
precluding the occurrences of extended ds-B-RNA helices but allowing more
open, A-type helices.
Jmol: Puckering in ribose and deoxyribose
Jmol:
Comparison of ds DNA forms
Jmol:
Comparison of ds DNA and RNA
Now lets review the kinds of structure adopted by the 3 major
macromolecules, DNA, RNA and proteins. DNA predominately adopts the
classic ds-BDNA structure, although this structure is wound around
nucleosomes and "supercoiled" in cells since it must be packed into the
nucleus. This extended helical form arise in part from the significant
electrostatic repulsions of two strands of this polyanions (even in the
presence of counterions). Given its high charge density, it is not surprising
that it is complexed with positive proteins and does not adopt complex
tertiary structures. RNA, on the other hand, can not form long B-type
double-stranded helices (due to steric constraints of the 2'OH and the
resulting 3'endo ribose pucker). Since it doesn't have the same charge
density as the double-stranded DNA, it can adopt complex tertiary
conformations (albeit with significant counterion binding to stabilize the
structure) and in doing so can form regions of secondary structure (ds-A
RNA) in the form of stem/hairpin forms. Proteins, with its combination of
polar charged, polar uncharged, and nonpolar side chains have little
electrostatic hindrance in the adoption of secondary and tertiary structures.
That RNA and proteins can both adopt tertiary structures with potential
binding and catalytic sites makes them ideal catalysts for chemical reactions.
RNA, given its 4 nucleotide motif can clearly also carry genetic information,
making it an ideal candidate for the first evolved macromolecules enabling
the development of life. Proteins with a great abundance of organic
functionalities would eventually supplant RNA as a better choice for life's
catalyst. DNA, with its greater stability, would supplant RNA as the choice for
the main carrier of genetic information.

RNA World Web Site
Recent References
1. Ben-Shem, A. et al. Crystal Structure of the Eukaryotic Ribosome.
Science 330, 1203 (2010)
2. Schmeing, T. and Ramakrishan, V. What recent ribosome structures
have revealed about the mechanism of translation. Nature 461, 1234
(2009)
3. Rodina, M. et al. How ribosomes make peptide bonds. TIBS 32, 20
(2007)
4. M. Simonović, T.A. Steitz, A structural view on the mechanism of the
ribosome-catalyzed peptide bond formation,Biochim. Biophys. Acta
(2009), doi:10.1016/j.bbagrm.2009.06.006
5. Shechner, David M., et al. "Crystal Structure of the Catalytic Core of an
RNA-Polymerase Ribozyme." Science 326, 1271-1275 (2009)
6. Doudna, J. & Lorsch, J. Ribozyme catalysis: Not different, just worse.
Nature Structural and Molecular Biology. 12, pg 395 (2005)
7. Adams, P. et al. Crystal structure of a self-splicing group I intron with
both exons. Nature. 430, pg 45 (2004)
8. Winkler, W. et al. Control of gene expression by a natural metaboliteresponsive ribozyme. Nature. 428, pg 281 (2004)
9. Rupert, P. et al. Transition State Stabilization by a Catalytic RNA.
Science, 298, pg 1421 (2002)
10. Rupert. P. & Ferré-D'Amaré, A. (2001) Crystal structure of a hairpin
ribozyme-inhibitor complex with implications for catalysis. Nature 410,
780-786 (2001)
11. Doudna and Cech. The chemical Repertoire of Natural Ribozymes.
Nature. 418, pg 222 (2002)
12. Shu-ichi Nakano et al. General Acid-Base Catalysis in the Mechanism of a
Hepatitis Delta Virus Ribozyme. 287,
Science 287, pg. 1493 (2000)
Delta Virus Ribozyme
13. Rupert and Ferre-D'Amare. The Hairpin turn (in ribozymes and their catalytic
mechanism). Nature. 410, pg 761 (2001)
14. Schultes and Bartel. One Sequence, Two Ribozymes: implications for the
emergence of new ribozyme folds. Science. 289, pg 401, 448 (2000)
15. Steitz at al. . The ribosome is a ribozyme. Science. 289, pg 878, 905 (2000)
16. Yean et al. The case for an RNA enzyme (Spliceosome) Nature. 408. pg 782,
881 (2000)
17. Perrota et al. Imidazole rescue of a Cytosine mutation in a self-cleaving
ribozyme. Science. 286. pg 61, 123 (2000)
18. Zhuang et al. A Single-Molecule Study of RNA Catalysis and Folding. Science.
288, pg 2048 (2000)
Navigation

Biochemistry Online: An Approach Based on Chemical Logic Table of Contents

Biochemistry BCHM 321: Home Page