trna Processing and Modification
RNA POL III - TRANSCRIPTS 5S RNA, trna, repetitive Sequenzen (Alu-typ), versch. kleine stabile RNAs (7SL - RNA vom signal recognition particle (SRP)), U6 RNA 5S RNA nicht wie bei Prokaryoten mit rrna s assoziiert. trna: 4.5S precursor Prozessieren 4S RNA Prokaryoten: 1 3 2 1 RNase P (Endonuklease) 2 Exonuklease 3 CCA- Addition trna nucleotidyltransferase
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 genes corresponding to 16 more modification and processing events remain to be 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)
Schematics of a trna precursor and a mature trna trna precursor mature trna Hopper, A. K. and Phizicky, E. M. Genes Dev. 17, 162-180 (2003)
trna 5 and 3 end processing pathways 5-15 nt CCA adding enzyme CCase Ntase Schurer, H. et al. Biol. Chem. 382, 1147-1156 (2001)
trna processing by RNase P
RNAse P carries out the 5 endonucleolytic cleavage of trnas in eubacteria, archaea, and eukaryotes (unprocessed trna) (negative control) (processed trna) (cleaved 5 fragment) M1 RNA from E. coli RNase P has enzymatic activity
3 end processing of trna
The CCA sequence the 3 end of trnas is essential for trna aminoacylation but is not encoded in nearly all eukaryotic trna genes Only three enzymes are known that add nucleotides to nucleic acid in a primer-dependent but template-independent fashion: 1. CCA-adding enzyme 2. poly(a) polymerase 3. terminal deoxynucleotidyltransferase (TdT)
trna splicing red-intron green-anticodon S. cerevisiae contains 272 trna genes of which 59 (encoding 10 different trnas) are interrupted by introns
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. Anticodon in vitro Reaktion: braucht ATP: 2 Reaktionen: Spaltung (-ATP) Ligation (+ATP)
Mechanism of trna splicing intron 1 cyclic phosphodiesterase adenylyl synthetase 2 ligase (2 phosphotransferase) 3 Abelson, J. et al. J. Biol. Chem. 273, 12685-12688 (1998)
Mechanism
trnas contain numerous modified nucleosides (amino acid) (dihydrouridine loop) (TψC loop) trna nucleosides are modified post-transcriptionally by multiple enzymatic systems invariant nucleotides are circled
Examples of modified nucleosides in trna Magenta represents the variation from one of the four normal RNA nucleosides
Three-dimensional structure of trna All trnas share a common cloverleaf secondary structure and a common tertiary structure which resembles an inverted L. The L shape maximizes stability by lining up base pairs in the D stem with those in the anticodon stem, and the base pairs in the T stem with those in the acceptor stem.
Tertiary interactions in trna
unusual base pairs increases stability of the trna structure
U ψ Blocking 2 -O-methylation or pseudouridine production at a global level in yeast rrna strongly inhibits growth rate and the absence of these modification in the U2 snrna impairs its assembly into an active spliceosomes in Xenopus oocytes.
Modification of nucleotides in trnas, rrnas snrnas, and snornas 1. 2 -O-methylation (modification of the ribose sugar) 2 -O-methyl transferase S-adenosylmethionine (SAM) CH 3 2. Conversion of uridine to pseudouridine (Ψ) by pseudouridine synthase
Eukaryots: 3 RNA - Polymerases POL I POL II POL III Lokalisation Nucleolus Nucleoplasm Nucleoplasm Product rrna hnrna, mrna 5S RNA, trna snrna 7S RNA % Cellactivity 50-70% 20-40% ~10% α-amanitin (50% Inhibition) Mammals > 400 µg/ml 0,025 µg/ml 20 µg/ml Yeast (S. Cerevisae)600 µg/ml 1µg/ml > 2000 µg/ml