Académique Documents
Professionnel Documents
Culture Documents
Prokaryotic Transcription
2. tRNA
3. rRNA
4. snRNA
CAPPING
Cap
structure on
the 5 end
of mRNA
molecules
Cap Functions
Cap provides:
1. Protection from some ribonucleases
2. Enhanced translation
3. Enhanced transport from nucleus
4. Enhances splicing of first intron for some
mRNAs
or RNA triphosphatase
Capping:
order of
events and
enzymes
AdoMet = S-adenosylmethionine,
the methyl donor
mGpppNmpNm
SPLICING
2
3
4
5
Eukaryotes
The occurrence of introns varies.
The majority of genes in vertebrates contain
introns.
genes encoding histones do not have introns.
Prokaryotes
Most prokaryotes do not have introns.
A few bacteria and archaebacteria have introns.
Splice Sites
Conserved splice sites are shared by
both the exon and the intron.
Different sequences on the donor site
(3) and on the acceptor site (5).
POLIADENYLATION
3 poly(A) tail
rRNA processing
Ribosome Structure
bacteria
vertebrates
Transcription rRNA
Processing
Role of small nucleolar RNAs
(snoRNAs)
Packaged with proteins to form
small nucleolar ribonucleoproteins
(snoRNPs)
snoRNPS associate with rRNA
before it is fully transcribed
Two groups of snoRNAs
U3 & antisense
tRNA processing
Synthesis of tRNA:
1.
2.
3.
4.
tRNAs form clover-leaf structures, with complementary basepairing between regions to form four stems and loops.
5.
6.
Ribozyme
Ribozymes
Discoveredin1980s
(Cech&Altman,
NobelPrize1989)
RNAcanactasanEnzyme
andcatalyseReactions
including
Itsownreplication
TheRNAWORLD
CatalyticMechanismofGroupIIntron:TomCechetal.
Hammerhead Ribozyme
Scott WG, Finch JT, Klug A. (1995) The crystal structure of an all-RNA hammerhead ribozyme: a proposed mechanism for RNA catalytic
cleavage. Cell 81, 991-1002.
Hammerhead ribozymes are catalytic RNAs found in plants and some pathogens. Their reactions are very limited, typically
strand cleavage. They are all metalloenzymes, usually using Mg. Several hammerhead structures have been solved. This one is
a minimized RNA which still retains catalytic activity; it has a 16 base enzyme strand and a 25 base substrate strand. In the
crystal, however, the usual cleavage site at C17, has been replaced with a non-hydrolyzable 2 deoxy nucleotide. The structure
shows a gamma shaped molecule, with stems I, II, and III flanking a conserved 16-base core which is required for structure
and activity. These core bases do not form Watson-Crick pairs, but a variety of adventitious interactions.
Hairpin ribozymes:
plant virus satellite RNAs mediate rolling circle
replication
Two main helical regions
G8 is essential but its role in catalysis
may be structural (this may be true for
metal ions as well)
G8
Active
A ??
is an
RNA satellite virus of hepatitis B virus
(HBV).
2.3
resolution
23S rRNA
A-site tRNA
P-site tRNA
RNA WORLD?
clinical
applications of Ribozymes
Emerging
4 main categories
1.
2.
3.
4.
Gene inhibitors
Gene amenders
Protein inhibitors
Immunostimulatory RNAs
Results
1.Transduced cells are well tolerated
and persist in the patient
2. Transduced cells may possess
transient survival advantage over
control cells
Shortcomings
fix p53 in
cancer cells?
Trans-splicing
mediated repair of
mutant transcripts
The concept
Exploit group I introns to cut and
paste exons in a directed fashion
A. The ribozyme is linked to
a W.T. gene fragment
B. Corrupt cis-splicing by
inserting a W.T. exon
SMaRT slpiceosome-mediated RNA trans-splicing (CFTR^F508)
Somemoderndaycoenzymes
maybetheevolutionaryremnants
ofmodifiednucleotidesin
catalyticRNA