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

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.

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
Discoveredin1980s
(Cech&Altman,
NobelPrize1989)
RNAcanactasanEnzyme
andcatalyseReactions
including
Itsownreplication

TheRNAWORLD

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)

CatalyticMechanismofGroupIIntron:TomCechetal.

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 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 ??

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
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 RNAs

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.

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

Three successive stages in

the evolution of a selfreplicating system of RNA
molecules capable of
directing protein synthesis.