trna processing and modification - eurasnet · in yeast, 38 genes are known to participate in trna...
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tRNA Processing and Modification
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RNA POL III - TRANSCRIPTS5S RNA, tRNA, repetitive Sequenzen (Alu-typ), versch. kleine stabile RNAs(7SL - RNA vom „signal recognition particle“ (SRP)), U6 RNA5S RNA nicht wie bei Prokaryoten mit rRNA‘s assoziiert.
tRNA: 4.5S precursor 4S RNAProzessieren
Prokaryoten:
21
3
1
RNase P (Endonuklease)
2
Exonuklease
3
CCA- Addition tRNA nucleotidyltransferase
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tRNA maturation requires five major steps
1. Removal of the 5’ leader sequence by RNase P
2. Removal of the 3’ trailer sequence by a combination of endonucleases
and exonucleases
3. Addition of CCA to the 3’ end
4. Splicing of introns in some tRNAs
5. Numerous modifications at multiple residues
6. Separation of multiple tRNAs
•In yeast, 38 genes are known to participate in tRNA processing and genescorresponding to 16 more modification and processing events remain tobe identified.•Thus, ~1% of the yeast genome is involved in tRNA processing
Hopper, A. K. and Phizicky, E. M. Genes Dev. 17, 162-180 (2003)
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tRNA precursor mature tRNA
Schematics of a tRNA precursor and a mature tRNA
Hopper, A. K. and Phizicky, E. M. Genes Dev. 17, 162-180 (2003)
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tRNA 5’ and 3’ end processing pathways
Schurer, H. et al. Biol. Chem. 382, 1147-1156 (2001)
5-15 nt
CCA adding enzymeCCaseNtase
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tRNA processing by RNase P
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RNAse P carries out the 5’ endonucleolytic cleavage oftRNAs in eubacteria, archaea, and eukaryotes
(negative control)(unprocessed tRNA)
(processed tRNA)
(cleaved 5’ fragment)
•M1 RNA from E. coli RNase P has enzymatic activity
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3’ end processing of tRNA
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The CCA sequence the 3’ end of tRNAs is essential for tRNAaminoacylation but is not encoded in nearly all eukaryotic tRNA genes
Only three enzymes are known that add nucleotides to nucleic acid in aprimer-dependent but template-independent fashion:1. CCA-adding enzyme2. poly(A) polymerase 3. terminal deoxynucleotidyltransferase (TdT)
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tRNA splicing
•S. cerevisiae contains 272 tRNA genes of which 59 (encoding 10 differenttRNAs) are interrupted by introns
red-introngreen-anticodon
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Eukaryotische tRNAs: Introns (auch Archebakterien)Hefe: Introns ca. 14 - 46 nt lang: Intronstruktur ist wichtig (A-I)ts Mutanten in Hefe →
precursor tRNA + w.t. Extrakt →
Spleißen. Hefe tRNA precursor auch von Xenopusextrakten prozessiert.
„in vitro“ Reaktion: braucht ATP: 2 Reaktionen: Spaltung (-ATP)Ligation (+ATP)
Anti-codon
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Mechanism of tRNA splicing
adenylylsynthetase
ligase
cyclic phosphodiesterase
intron
Abelson, J. et al. J. Biol. Chem. 273, 12685-12688 (1998)
(2’ phosphotransferase)
1
2
3
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Mechanism
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tRNAs contain numerous modified nucleosides
•tRNA nucleosides are modified post-transcriptionally by multiple enzymatic systems•invariant nucleotides are circled
(amino acid)
(dihydrouridine loop)
(TψC loop)
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Examples of modified nucleosides in tRNA
Magenta represents the variation from one of the four normal RNA nucleosides
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Three-dimensional structure of tRNA
•All tRNAs share a common cloverleaf secondary structure and a commontertiary structure which resembles an inverted L.•The L shape maximizes stability by lining up base pairs in the D stem withthose in the anticodon stem, and the base pairs in the T stem with those inthe acceptor stem.
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Tertiary interactions in tRNA
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•unusual base pairs increases stability of the tRNA structure
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•Blocking 2’-O-methylation or pseudouridine production at a global levelin yeast rRNA strongly inhibits growth rate and the absence of thesemodification in the U2 snRNA impairs its assembly into an activespliceosomes in Xenopus oocytes.
U
ψ
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Modification of nucleotides in tRNAs, rRNAssnRNAs, and snoRNAs
CH3
S-adenosylmethionine (SAM)
2’-O-methyl transferase
1. 2’-O-methylation (modification of the ribose sugar)
2. Conversion of uridine to pseudouridine (Ψ) by pseudouridine synthase
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Eukaryots: 3 RNA - Polymerases
POL I POL II POL IIILokalisation Nucleolus Nucleoplasm Nucleoplasm
Product rRNA hnRNA, mRNA 5S RNA, tRNAsnRNA 7S RNA
% Cellactivity 50 - 70% 20 - 40% ~10%
α-Amanitin (50% Inhibition)Mammals > 400 µg/ml 0,025 µg/ml 20 µg/mlYeast (S. Cerevisae)600 µg/ml 1µg/ml > 2000 µg/ml