Eukaryotic vs. Prokaryotic TranscriptionIn 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 polypeptidemoncistronic: 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: mRNAMessenger RNA, encodes the amino acid sequence of a polypeptide2. tRNATransfer RNA, transports amino acids to ribosomes during translation3. rRNARibosomal RNA, forms complexes called ribosomes with protein, the structure on which mRNA is translated4. snRNASmall nuclear RNA, forms complexes with proteins used in eukaryotic RNA processing

Initial gene products = precursor RNAs (3 main classes = pre-rRNA, pre-tRNA, pre-mRNA)All precursor RNAs are shortened and modified. Shortening can either be by nucleases or splicing. Modifications = addition of nucleotides (e.g. polyA tails) and chemical modification. Main kinds of modified nucleotides are 7-methyl-guanosine-5'ppp5'NNN cap (mRNA) and 2'-O-methyl and pseudouridine groups (stable RNAs).

Potential Steps for Regulation of Eukaryotic Gene Expression

Steps in mRNA processing (hnRNA is the precursor of mRNA) capping (occurs co-transcriptionally) cleavage and polyadenylation (forms the 3 end) splicing (occurs in the nucleus prior to transport)

CAPPING

Cap structure on the 5 end of mRNA molecules

Cap FunctionsCap provides:Protection from some ribonucleasesEnhanced translationEnhanced transport from nucleusEnhances splicing of first intron for some mRNAs

AdoMet = S-adenosylmethionine, the methyl donorCapping: order of events and enzymesProduct is Cap 1 (another Met at 3rd nucl =>Cap2)or RNA triphosphatase

All RNAs synthesized by RNA pol II are capped in one way or another.

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 nucleotidespppNpNmGpppNmpNm

Formation of the 5' cap requires several steps, the first taking place immediately following initiation oftranscription. The initiating nucleotide has a 5' triphosphate end, which is first hydrolyzed to a monophosphateend and PPi. Guanylyltransferase then transfers a G residue to that monophosphate end, using GTP as thesubstrate, releasing PPi. The bond that is formed is a 5'-5' triphosphate bond. Methyltransferases thenmethylate the terminal guanosine on the 7 position, forming 7-methyl guanosine, and methylate the next twonucleotides at the 2' ribose positions.

SPLICING

Overview of the processing of a eukaryotic mRNA

RNA splicing is the removal of intervening sequences (IVS) that interrupt the coding region of a geneExcision of the IVS (intron) is accompanied by the precise ligation of the coding regions (exons)

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EukaryotesThe 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 intronsProkaryotesMost prokaryotes do not have introns.A few bacteria and archaebacteria have introns.

Splice SitesConserved 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

Polyadenylation: cleavage of the primary transcript occurs approximately 10-30 nucleotides 3-ward of the AAUAAA consensus site polyadenylation catalyzed by poly(A) polymerase approximately 200 adenylate residues are added

rRNA processing

Ribosome Structure

S = Svedberg, a measure of sedimentation in centrifuge

Transcription rRNA Processing

Ribosomal RNA GenesTandemly repeatedNon-transcribed spacers45S rRNA precursor 18S, 5.8S, 28S rRNA

Synthesis of rRNA occurs within nucleolus. The nucleolus is a nuclear suborganelle produced at sites of rRNA genes through the action of nucleolar organizer associated with the rRNA gene locus (requires only a single rRNA gene for nucleolus formation). Nucleoli are the sites for synthesis, modification, processing, and assembly of rRNA into ribosomes. Nucleoli may form containing rRNA genes derived from multiple different chromosomes in humans.

rRNA PROCESSINGSmall eukaryotic subunit of the ribosome (the so-called 40S subunit) contains:18S rRNA and associated proteinsLarge eukaryotic subunit (60S) contains:28S, 5.8S, and 5S rRNA and associated proteinsThe main rRNA locus in eukaryotes consists of the 28S-5.8S-18S genes linked together. The linked genes are tandemly repeated with regions surrounding the genes known as ETS, ITS, and NTS(the NTS is critical for initiation and termination of Tx via pol I)A large 45S pre-rRNA transcript is formed consisting of ETS -- 18S -- ITS -- 5.8S -- ITS -- 28S -- ETS

Processing of Human 45S rRNA Precursor

Processing Bacterial rRNA Precursor

bacteria

vertebrates

RNA modification: snoRNASmall 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 ProcessingRole of small nucleolar RNAs (snoRNAs)Packaged with proteins to form small nucleolar ribonucleoproteins (snoRNPs)snoRNPS associate with rRNA before it is fully transcribedTwo groups of snoRNAsU3 & antisenseAntisense forms an RNA duplexRecognition site for enzymes which modify pre-RNA

snoRNAs also involved in rRNA maturationFunctions, Roles, or Types of snoRNAs1. Direct sites for 2'-0-methylation of ribose in rRNA maturation2. Direct sites for pseudouridine formation in rRNA maturation3. Direct pre-rRNA cleavage and correct structure formation for rRNA maturation

tRNA processing

Synthesis of tRNA:

tRNA genes also occur in repeated copies throughout the genome, and may contain introns.

Each tRNA (75-90 nt in length) has a different sequence that binds a different amino acid.

Many tRNAs undergo extensive post-transcription modification, especially those in the mitochondria and chloroplast.

tRNAs form clover-leaf structures, with complementary base-pairing between regions to form four stems and loops.

Loop #2 contains the anti-codon, which recognizes

mRNA codons during translation.

Same general mechanism using RNA polymerase III, promoters, unique TFs, plus posttranscriptional modification from pre-tRNA.

tRNA molecules are transcribed as longer precursor molecules which are cleaved and chemically modified. RNase P processes all tRNA 5'-termini and has a catalytically active RNA moiety. (RNase D processes 3'-ends. ) CCA addition occurs at 3'-end of all tRNAs. Bases and ribose are chemically modified. Many unusual bases are found in tRNA. tRNA maturation in some eucaryotes can require 5'-leader sequence cleavage, intron removal (splicing), replacement of 3'-UU with 3'-CCA, RNA editing, and several base modifications.

Sphere = cleavage pointRNase 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 BasesCreated 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.

RibozymesDiscovered in 1980s(Cech & Altman, Nobel Prize 1989)RNA can act as an Enzyme and catalyse Reactions includingIts own replicationThe RNA WORLD

the discovery two decades ago that certain RNAs perform catalytic reactions tumbled the molecular dogma that all enzymes are proteins.

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)

Ribozymes, like traditional protein enzymes, act to lower activation energies and stabilize reaction intermediates, however RNA lacks the functional amino acid side chains that can readily participate in acid/base chemistry. That is, at a neutral pH, nucleosides seem to lack functional groups that could remove a proton from an attacking nucleophile or donate a proton to the 5 oxygen leaving group.Until recently, all catalytic RNAs were thought to solve this dilemma by recruiting metal cations and though this does not always seem to be an aboslute recuirment the role cations play in RNA catalysis is still the subject of ongoing research. All RNAs require cations to neutralize negatively charged phosphates during assembly of compact structures, so it can be difficult to distinguish a role for metal cations in RNA folding from direct participation of metal cations in catalytic chemistry.The significance of this discovery is most appeart by looking back on the current state of molecualr biology during the time of this discovery

Catalytic Mechanism of Group I Intron: Tom Cech et al.

RNA base pairs with an IGS (P1 helix). Exogenous guanine binds to a G site within P7 3-OH of the guanine acts as a nucleophile, attaching a polarized phosphate at the splice site, resulting in an inversion and release of the products. Transesterification reaction requires: monovalent cation, divalent cation and an exogenous guanine nucleotide

Group II introns: found in bacteria and organellar genes of eukaryotic cellsNucleophilic attack by the 2-OH of an adenosine within the branch siteConformational change releases the intron lariat and the mature exonGroup II introns encode proteins involved insplicing, integration and RT, promoting intron mobility

Hammerhead RibozymeScott 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 cellsSite specific hydrolysis of tRNA, 5S rRNA and signal recognition particle RNATwo domain structure Substrate recognitionRibozyme active site

Structural predictions of the RNA are made by doing phylogenetic comparisons

Hairpin ribozymes: plant virus satellite RNAs mediate rolling circle replicationTwo main helical regionsG8 is essential but its role in catalysis may be structural (this may be true for metal ions as well)

G8Active A ??

4 helix hairpin structure is stableG8 electrostatic stabilization of the substrate cleavage site phosphdiester through the N2 exocyclic amine and possible positioning of activation of the 2-OH by the N1

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 resolution

The RIBOSOME is a RIBOZYME

5S rRNA23S rRNAA-site tRNAP-site tRNAP AAdenine is highly conserved, participates in general acid-base catalysis to deprotonate the amineRibosomal proteins act as scaffolding to orient the catalytic RNARNA WORLD?

Emerging clinical applications of Ribozymesan alternative or in combination with siRNA

Gene inhibitorsGene amendersProtein inhibitorsImmunostimulatory RNAs4 main categories

Clinical applications for trans-cleaving ribozymes1.Transduced cells are well tolerated and persist in the patient 2. Transduced cells may possess transient survival advantage over control cellsResultsShortcomings Long term efficacy is still a question. Efficient gene delivery system.

Chem. hammerheadRIBOZYME clinical trials in progress: anti-cancer (VEGF) and anti-viral (HIV)

Trans-splicing mediated repair of mutant transcriptsThe conceptExploit group I introns to cut and paste exons in a directed fashion A. The ribozyme is linked to a W.T. gene fragmentB. Corrupt cis-splicing by inserting a W.T. exon fix p53 in cancer cells?SMaRT slpiceosome-mediated RNA trans-splicing (CFTR^F508)

The capabilities as well as the function and role of RNA molecules in cellular processes suggest that early life forms may have been RNA based and that even perhaps the first or early life forms may have evolved from an "RNA World" = RNA WORLD HYPOTHESIS

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.

Initial gene products = precursor RNAs (3 main classes = pre-rRNA, pre-tRNA, pre-mRNA)All precursor RNAs are shortened and modified. Shortening can either be by nucleases or splicing. Modifications = addition of nucleotides (e.g. polyA tails) and chemical modification. Main kinds of modified nucleotides are 7-methyl-guanosine-5'ppp5'NNN cap (mRNA) and 2'-O-methyl and pseudouridine groups (stable RNAs).Steps in mRNA processing (hnRNA is the precursor of mRNA) capping (occurs co-transcriptionally) cleavage and polyadenylation (forms the 3 end) splicing (occurs in the nucleus prior to transport)

All RNAs synthesized by RNA pol II are capped in one way or another.Formation of the 5' cap requires several steps, the first taking place immediately following initiation oftranscription. The initiating nucleotide has a 5' triphosphate end, which is first hydrolyzed to a monophosphateend and PPi. Guanylyltransferase then transfers a G residue to that monophosphate end, using GTP as thesubstrate, releasing PPi. The bond that is formed is a 5'-5' triphosphate bond. Methyltransferases thenmethylate the terminal guanosine on the 7 position, forming 7-methyl guanosine, and methylate the next twonucleotides at the 2' ribose positions.RNA splicing is the removal of intervening sequences (IVS) that interrupt the coding region of a geneExcision of the IVS (intron) is accompanied by the precise ligation of the coding regions (exons)Polyadenylation: cleavage of the primary transcript occurs approximately 10-30 nucleotides 3-ward of the AAUAAA consensus site polyadenylation catalyzed by poly(A) polymerase approximately 200 adenylate residues are added

Synthesis of rRNA occurs within nucleolus. The nucleolus is a nuclear suborganelle produced at sites of rRNA genes through the action of nucleolar organizer associated with the rRNA gene locus (requires only a single rRNA gene for nucleolus formation). Nucleoli are the sites for synthesis, modification, processing, and assembly of rRNA into ribosomes. Nucleoli may form containing rRNA genes derived from multiple different chromosomes in humans.rRNA PROCESSINGSmall eukaryotic subunit of the ribosome (the so-called 40S subunit) contains:18S rRNA and associated proteinsLarge eukaryotic subunit (60S) contains:28S, 5.8S, and 5S rRNA and associated proteinsThe main rRNA locus in eukaryotes consists of the 28S-5.8S-18S genes linked together. The linked genes are tandemly repeated with regions surrounding the genes known as ETS, ITS, and NTS(the NTS is critical for initiation and termination of Tx via pol I)A large 45S pre-rRNA transcript is formed consisting of ETS -- 18S -- ITS -- 5.8S -- ITS -- 28S -- ETS

snoRNAs also involved in rRNA maturationFunctions, Roles, or Types of snoRNAs1. Direct sites for 2'-0-methylation of ribose in rRNA maturation2. Direct sites for pseudouridine formation in rRNA maturation3. Direct pre-rRNA cleavage and correct structure formation for rRNA maturation

tRNA molecules are transcribed as longer precursor molecules which are cleaved and chemically modified. RNase P processes all tRNA 5'-termini and has a catalytically active RNA moiety. (RNase D processes 3'-ends. ) CCA addition occurs at 3'-end of all tRNAs. Bases and ribose are chemically modified. Many unusual bases are found in tRNA. tRNA maturation in some eucaryotes can require 5'-leader sequence cleavage, intron removal (splicing), replacement of 3'-UU with 3'-CCA, RNA editing, and several base modifications.

the discovery two decades ago that certain RNAs perform catalytic reactions tumbled the molecular dogma that all enzymes are proteins. Ribozymes, like traditional protein enzymes, act to lower activation energies and stabilize reaction intermediates, however RNA lacks the functional amino acid side chains that can readily participate in acid/base chemistry. That is, at a neutral pH, nucleosides seem to lack functional groups that could remove a proton from an attacking nucleophile or donate a proton to the 5 oxygen leaving group.Until recently, all catalytic RNAs were thought to solve this dilemma by recruiting metal cations and though this does not always seem to be an aboslute recuirment the role cations play in RNA catalysis is still the subject of ongoing research. All RNAs require cations to neutralize negatively charged phosphates during assembly of compact structures, so it can be difficult to distinguish a role for metal cations in RNA folding from direct participation of metal cations in catalytic chemistry.The significance of this discovery is most appeart by looking back on the current state of molecualr biology during the time of this discoveryRNA base pairs with an IGS (P1 helix). Exogenous guanine binds to a G site within P7 3-OH of the guanine acts as a nucleophile, attaching a polarized phosphate at the splice site, resulting in an inversion and release of the products. Transesterification reaction requires: monovalent cation, divalent cation and an exogenous guanine nucleotide4 helix hairpin structure is stableG8 electrostatic stabilization of the substrate cleavage site phosphdiester through the N2 exocyclic amine and possible positioning of activation of the 2-OH by the N1

The capabilities as well as the function and role of RNA molecules in cellular processes suggest that early life forms may have been RNA based and that even perhaps the first or early life forms may have evolved from an "RNA World" = RNA WORLD HYPOTHESIS