tRNA Processing and Modification

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

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)

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)

tRNA 5’ and 3’ end processing pathways

Schurer, H. et al. Biol. Chem. 382, 1147-1156 (2001)

5-15 nt

CCA adding enzymeCCaseNtase

tRNA processing by RNase P

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

3’ end processing of tRNA

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)

tRNA splicing

•S. cerevisiae contains 272 tRNA genes of which 59 (encoding 10 differenttRNAs) are interrupted by introns

red-introngreen-anticodon

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

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

Mechanism

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)

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 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.

Tertiary interactions in tRNA

•unusual base pairs increases stability of the tRNA structure

•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

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

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