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

AdoMet = S-adenosylmethionine, the methyl donor

Capping: order of events and enzymes

Product is Cap 1 (another Met at 3rd nucl =>Cap2)

or “RNA triphosphatase”

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 onthe next two nucleotides

pppNpN

mGpppNmpNm

SPLICING

Overview of the processing of a eukaryotic mRNA

1

23

45

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

3’ poly(A) tail

Cleavage signal sequence

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 base-pairing 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

RNase P RNA forms a pocket around the pre-tRNA, creating an enzymaticly favorable environment for cleavage.

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.

Function of Unusual Bases

• Created post-transcriptionally.

• Purpose is sometimes to allow for promiscuous base-pairing: 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 includingIts 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)

• Nested double pseudoknot in which the active site is buried (100-fold faster the Hammerheads)

2.3 Å resolution

The RIBOSOME is a RIBOZYMEThe RIBOSOME is a RIBOZYME5S rRNA

23S rRNA

A-site tRNA

P-site tRNA

PP AA

Adenine is highly conserved, participates in general acid-base catalysis to deprotonate the amine

Ribosomal proteins act as scaffolding to orient the catalytic RNA

RNA WORLD?

EmergingEmerging clinical clinical applications of Ribozymesapplications of Ribozymes

an alternative or in combination with siRNA

1. Gene inhibitors

2. Gene amenders

3. Protein inhibitors

4. Immunostimulatory RNA’s

4 main categories

Clinical applications for Clinical applications for trans-cleaving ribozymestrans-cleaving ribozymes

1.Transduced cells are well tolerated and persist in the patient

2. Transduced cells may possess transient survival advantage over control cells

Results

Shortcomings 1. Long term efficacy is still a question.

2. Efficient gene delivery system.

Chem. Chem. hammerheadhammerhead

RIBOZYME clinical trials in progress: RIBOZYME clinical trials in progress: anti-cancer anti-cancer (VEGF)(VEGF) and anti-viral and anti-viral (HIV)(HIV)

TransTrans-splicing -splicing mediated repair of mediated repair of mutant transcriptsmutant transcripts

The conceptThe 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

fix p53 in fix p53 in cancer cells?cancer cells?

SMaRT slpiceosome-mediated RNA trans-splicing (CFTR^F508)SMaRT slpiceosome-mediated RNA trans-splicing (CFTR^F508)

Some modern day coenzymesmay be the evolutionary remnantsof modified nucleotides in catalytic RNA

Three successive stages in the evolution of a self-

replicating system of RNA molecules capable of

directing protein synthesis.