Transcript Slide 1

Eukaryotic vs. Prokaryotic Transcription
• In eukaryotes, transcription and translation occur in separate
compartments.
• In bacteria, mRNA is polycistronic; in eukaryotes, mRNA is usually
monocistronic.
– Polycistronic: one mRNA codes for more than one polypeptide
– moncistronic: one mRNA codes for only one polypeptide
• 3 RNA polymerases in euk., 1 in prok.
• “Processing” of mRNA in eukaryotes, no processing in prokaryotes
Four different types of RNA, each encoded by different genes:
1. mRNA
Messenger RNA, encodes the amino acid sequence
of a polypeptide
2. tRNA
Transfer RNA, transports amino acids to ribosomes
during translation
3. rRNA
Ribosomal RNA, forms complexes called ribosomes
with protein, the structure on which mRNA is
translated
4. snRNA
Small nuclear RNA, forms complexes with proteins
used in eukaryotic RNA processing
Potential Steps for Regulation of
Eukaryotic Gene Expression
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
Product is Cap 1 (another Met at 3rd nucl =>Cap2)
Capping occurs co-transcriptionally shortly after initiation
• guanylyltransferase (nuclear) transfers G residue to 5’ end
• methyltransferases (nuclear and cytoplasmic) add methyl
groups to 5’ terminal G and at two 2’ ribose positions on
the next two nucleotides
pppNpN
mGpppNmpNm
SPLICING
Overview of the processing of a
eukaryotic mRNA
1
2
3
4
5
6
7
• Eukaryotes
– The occurrence of introns varies.
• The majority of genes in vertebrates contain
introns.
– genes encoding histones do not have introns.
• The yeast have many genes that lack 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
Cleavage signal sequence
3’ poly(A) tail
rRNA processing
Ribosome Structure
S = Svedberg, a measure of sedimentation in centrifuge
• Transcription – rRNA Processing
Ribosomal RNA Genes
• Tandemly
repeated
• Non-transcribed
spacers
• 45S rRNA
precursor → 18S,
5.8S, 28S rRNA
Processing of Human 45S rRNA
Precursor
Processing Bacterial rRNA
Precursor
bacteria
vertebrates
RNA modification: snoRNA
• Small nucleolar RNA (snoRNA) has many
modifying functions including methylation
and pseudouridylation of pre-rRNA.
• The exact purpose of these modifications
are still unknown except to say that they
somehow guide the rRNA subunits to form
a functional ribosome.
• 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
• Antisense forms an RNA duplex
– Recognition site for enzymes which
modify pre-RNA
tRNA processing
Synthesis of tRNA:
1.
tRNA genes also occur in repeated copies throughout the genome,
and may contain introns.
2.
Each tRNA (75-90 nt in length) has a different sequence that
binds a different amino acid.
3.
Many tRNAs undergo extensive post-transcription modification,
especially those in the mitochondria and chloroplast.
4.
tRNAs form clover-leaf structures, with complementary basepairing between regions to form four stems and loops.
5.
Loop #2 contains the anti-codon, which recognizes
mRNA codons during translation.
6.
Same general mechanism using RNA polymerase III, promoters,
unique TFs, plus posttranscriptional modification from pre-tRNA.
Ribozyme
Sphere = cleavage point
Ribonuclease P (RNase P) is a
holoenzyme that cleaves the 5¢
leader element of pre-tRNA to
produce mature tRNA.
It is found in all domains of life as well
as mitochondria and chloroplasts.
RNase P is made of a catalytically
active ncRNA component and protein
component.
RNase P RNA forms a pocket
around the pre-tRNA, creating an
enzymaticly favorable
environment for cleavage.
Function of Unusual Bases
• Created post-transcriptionally.
• Purpose is sometimes to allow for promiscuous basepairing: Inosine in the 1st “wobble” position of
anticodon can bind to 3rd U, C or A in codon.
• This means that fewer different tRNAs are required.
• Others play a structural role.
Ribozymes
Discovered in 1980’s
(Cech & Altman,
Nobel Prize 1989)
RNA can act as an Enzyme
and catalyse Reactions
including
Its own replication
The RNA WORLD
Enzymes function to reduce the
activation energy
Reactions catalyzed by RNA can be characterized in the same way
as classical protein enzyme reactions (Michaelis-Menten kinetics: 103-106)
Catalytic Mechanism of Group I Intron: Tom Cech et al.
Group II introns: found in
bacteria and organellar genes of eukaryotic cells
1. Nucleophilic attack by the
2’-OH of an adenosine
within the branch site
2. Conformational change
releases the intron lariat
and the mature exon
Group II introns encode proteins involved in
splicing, integration and RT, promoting intron mobility
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.
Ribonuclease P: found in all cells
• Site specific hydrolysis of tRNA, 5S rRNA
and signal recognition particle RNA
• Two domain structure
– Substrate recognition
– Ribozyme active site
Structural predictions of the RNA are
made by doing phylogenetic
comparisons
Hairpin ribozymes:
plant virus satellite RNA’s 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 ??
Hepatitis delta virus:
is an RNA
satellite virus of hepatitis B virus (HBV).
• HDV is the only catalytic RNA known to be
required for the viability of a human
pathogen and is the fastest known naturally
occurring self-cleaving RNA
(first-order rate of 52 reactions/min)
2.3 Å resolution
• Nested double pseudoknot in
which the active site is buried
(100-fold faster the Hammerheads)
The RIBOSOME is a RIBOZYME
5S rRNA
P A
Ribosomal proteins act
as scaffolding to orient
the catalytic RNA
23S rRNA
A-site tRNA
P-site tRNA
Adenine is highly conserved,
participates in general acidbase catalysis to deprotonate
the amine
RNA WORLD?
clinical
applications of Ribozymes
Emerging
an alternative or in combination with siRNA
4 main categories
1.
2.
3.
4.
Gene inhibitors
Gene amenders
Protein inhibitors
Immunostimulatory
RNA’s
Clinical applications for
trans-cleaving ribozymes
RIBOZYME clinical trials in progress:
anti-cancer (VEGF) and anti-viral (HIV)
Chem.
hammerhead
Results
1.Transduced cells are well tolerated
and persist in the patient
2. Transduced cells may possess
transient survival advantage over
control cells
Shortcomings
1. Long term efficacy is still a question.
2. Efficient gene delivery system.
Trans-splicing
mediated repair of
mutant transcripts
fix p53 in
cancer cells?
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)
Some modern day coenzymes
may be the evolutionary remnants
of modified nucleotides in
catalytic RNA
Three successive stages in
the evolution of a selfreplicating system of RNA
molecules capable of
directing protein
synthesis.