Transcript orange messenger

Catalytic RNAs, riboswitches and
nucleoprotein complexes
The role of RNA in the cellular
environment is more complex than
originally thought.
This is thought to be due to the facts
that
–RNA can adopt complex 3D structures
–RNA can bind small molecule ligands
with high affinity and specificity and this
can lead to conformation changes
–RNA active sites can catalyse
biochemical transformations
RNAs role as a ribozyme (RNA enzymes)
Ribozymes are antisense RNA molecules that have catalytic activity
• Enzymes made of protein are the dominant form of biocatalyst in
modern cells
• There are at least eight natural RNA enzymes, or ribozymes, that
catalyze fundamental biological processes. It is believe that these
ribozymes might be the remnants of an ancient form of life that was
guided entirely by RNA.
• Discovered by Thomas Czech, he got the Noble prize for it in 1989. The
first ribozymes were discovered in Tetrahymena
The structure of RNA indicates the potential to be catalytic.
• It has the flexibility to fold into a three-dimensional, globular-like
structure which can bring potential catalytic groups into close contact
with the "substrate".
• Both the phosphate backbone, the 2'-OH and the base have potential
acid/base/nucleophile catalytic groups,
• and metal ions bound to the phosphate can be electrophilic.
RNA often controls the expression of genes, a role that had been thought to
be at least mostly the domain of proteins called "repressors" and
"transcription factors."
A number of labs around the world are now using these ribozymes to
study gene function
e.g. in the study of HIV, the AIDS virus, and in Cancer research.
Mang Yu and coworkers at the University of California recently used
ribozymes to provide white blood cells with resistance to HIV
infection.
A laboratory in Strasbourg is currently making use of ribozymes to
study the involvement of certain gene sequences in the onset of
Spinal Muscular Atrophy
They designed ribozymes that target the RNA messenger of certain
genetic sequences in muscle cells that have been cultured under
special conditions and observed for cellular changes similar to those
in diseased cells.
Hopefully this will not only lead to a larger understanding of exactly
what role such genetic sequences play in the onset of the disease
but may also allow new potential therapies to be developed.
Examples of Ribozymes in bacterial cells and viruses
1.
Plant viruses
•
Circular ribozymes, called viroids, have been discovered which can have
a devastating effect on plants.
•
The ribozymes replicate themselves in copies attached to their own
genome.
•
The viroids then undergo self-cleavage, sending fragments off to colonize
other areas of the plant.
•
The viroids harm the plants by rapidly proliferating and using nucleotide
materials the plant itself needs.
•
Further damage is caused as the viroid bundles interfere with the plant’s
internal structures much like a tumor.
•
They have no protein producing capabilities.
•
Their cleavage from the mother strand is completely self-controlled and
initiated.
•
They are catalytic in their own replication and processing.
•
A site is less than 30 nucleotides long and consists of three stems
coming off a central loop is critical.
•
This secondary structure, called a "hammerhead", is capable of cleaving
very specific sequences of RNA in order to release viable daughter
strands of RNA.
•
Synthetic hammerheads, consisting of only 19 nucleotides, have already
been produced, which can act as highly specific catalysts.
•
Similar synthetic ribozymes are being designed to break up RNA viruses
and RNA involved in the transcription and translation of mutant DNA.
Ribozyme examples cont.
2. ribonuclease P (RNase P)- function to cleave a piece of RNA off a
tRNA molecule
RNase P is able to selectively cut more than 60 tRNA precursors,
which then become mature tRNA molecules capable of carrying
amino acids during the translation of proteins.
.RNase P catalyzes specific phosphodiester bond hydrolysis in pretRNAs to produce mature tRNA 5'-ends.
Without RNase P this process would not be possible.
The enzyme is a ribonucleoprotein, although the RNA segment of the
molecule has been shown to independently recognize and cleave
the appropriate substrate both in vivo and in vitro.
The protein segment of the RNase P appears to allow the ribozymal
segment to work at a faster hydrolytic rate and with less Mg2+
present.
Ribozyme examples continued
3. The ribosome
the atomic-level picture of the ribosome
shows the complex fold of the RNA
molecules buttressed and supported by
numerous proteins.
the peptidyltransferase center is composed
of RNA, with no proteins in the vicinity
This enzymatic activity is due to the RNA
.
The active site of the ribosome
consists of RNA (white strands), not
protein (orange)
Riboswitches
• The switching on and switching off of genes in response to an
organism's needs is one of the most basic of biological control
mechanisms.
• Recently RNA elements built into messenger RNAs have been
found to directly sense the concentration of small metabolites and
turn gene expression on or off in response.
• These riboswitches fold into intricate structures that can distinguish
one metabolite from another .
• Three distinct tricks for switching gene expression have been
revealed:
• the RNA element can cause premature termination of transcription
of the mRNA,
• it can block ribosomes from translating the mRNA,
• or it can even cleave the mRNA and thereby promote its destruction.
This involves an RNA unit directly binding a small-molecule
metabolite, which switches the RNA into a conformation that
activates its intrinsic self-cleavage activity. This "ribozyme
riboswitch" represents a new type of biological activity for a catalytic
RNA.
A riboswitch (A) binds to its target molecule (B) and inhibits nearby
transcription machinery (C).
Riboswitches continued.
• Aptamers are RNA or DNA molecules selected
in vitro from vast populations of random
sequence that recognize specific ligands by
forming binding pockets.
• Allosteric ribozymes are RNA enzymes whose
activity is modulated by the binding of an effector
molecule to an aptamer domain, which is
located apart from the active site.
• These RNAs act as precision molecular switches
that are controlled by the presence or absence
of a specific effector.
Riboswitches continued
• Many antibiotics bind to ribosomal RNAs and selectively inhibit
bacterial growth.
• Riboswitches might also be targeted by new classes of antibiotics.
• Given the significant role that riboswitches play in bacterial genetic
control and the fact that they have evolved to bind metabolites, drug
compounds could be created that disrupt bacterial genetic control.
• Engineered riboswitches might function as designer genetic control
elements.
Natural Riboswitch Targets include:
• Coenzyme B12
• Thiamine pyrophosphate
• FMN
• S-adenosylmethionine
• Guanine
• Adenine
• Lysine
RNA Interference- another role for RNA
• double-stranded RNA (dsRNA) is a potent regulator of gene
expression .
• Cells maintain a multi-step pathway for dealing with dsRNAs, either
endogenous (those made by transcription of their own genes) or
exogenous.
• An enzyme called "dicer" cuts the dsRNAs into 20-base pair
fragments.
• One of the two strands is then transferred to a matching sequence
on a messenger RNA, and an enzyme called "slicer" then cleaves
the mRNA at the position of the duplex.
• The cleaved mRNA is rapidly degraded.
• In other cellular systems, instead of the mRNA being degraded it
stays intact, but the presence of the short RNA duplex renders it
somehow untranslatable, so no protein product is made.
• The discovery of RNA interference (RNAi) has led to the
identification of many small cellular RNAs that do not encode
proteins but instead act to regulate the expression of other genes .
• These microRNAs form extensively base-paired "foldback"
structures that are then processed by RNAi.
• RNAi has become a powerful tool for understanding which genes
are important for which biological events.