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Schlagwörter:
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Zusammenfassung:
Removal of internal transcribed spacer 2 (ITS2) from pre-ribosomal RNA
is essential to make functional ribosomes. This complicated processing
reaction begins with a single endonucleolytic cleavage followed by
exonucleolytic trimming at both new cleavage sites to generate mature
5.8S and 25S rRNA. We reconstituted the 7S -> 5.8S processing branch
within ITS2 using purified exosome and its nuclear cofactors. We find
that both Rrp44's ribonuclease activities are required for initial RNA
shortening followed by hand over to the exonuclease Rrp6. During the in
vitro reaction, ITS2-associated factors dissociate and the underlying
'foot' structure of the pre-60S particle is dismantled. 7S pre-rRNA
processing is independent of 5S RNP rotation, but 26S -> 25S trimming is
a precondition for subsequent 7S -> 5.8S processing. To complete the in
vitro assay, we reconstituted the entire cycle of ITS2 removal with a
total of 18 purified factors, catalysed by the integrated activities of
the two participating RNA-processing machines, the Las1 complex and
nuclear exosome.
