PRERIBOSOMAL RNA PROCESSING IN ARCHAEA - CHARACTERIZATION OF THE RNP ENDONUCLEASE MEDIATED PROCESSING OF PRECURSOR 16S RIBOSOMAL-RNA IN THETHERMOACIDOPHILE SULFOLOBUS-ACIDOCALDARIUS

The hyperthermophilic archaeon Sulfolobus acidocaldarius uses a novel RNA-containing endonuclease to excise and mature 16S rRNA from the pre cursor (pre) rRNA transcript. A cell-free processing system has been d eveloped using an in vitro transcribed RNA substrate containing the en tire 144 nucleotide 5' external transcribed spacer (5'ETS) and the fir st 72 nucleotides of 16S rRNA. The cell-free extract cleaves in the 5' ETS at positions -99, -31, and +1 (i.e., the 5'ETS-16S junction). Thes e positions are at nor near the positions cleaved in vivo during proce ssing of the pre rRNA transcript. The processing activity has been pur ified between 100 and 200-fold and appears to contain five or six poly peptide components and perhaps as many as 10 different small RNA compo nents. Using combined reverse transcription-PCR amplification, full or partial cDNA copies of two of the RNA components have been obtained. One of the RNAs exhibits sequence and structural similarities to eukar yotic U3 snoRNA. The processing activity has been shown to be inactiva ted by micrococcal nuclease. It can be reactivated by reconstituting u sing bulk RNA from S. acidocaldarius but not bulk RNA from distantly r elated organisms. The activity is also abolished by RNase H digestion in the presence of oligonucleotides complementary to the U3-like RNA. These results demonstrate that the U3-like RNA is an essential compone nt of the pre rRNA processing RNP endonuclease. Furthermore, this RNP endonuclease is not a derived eukaryotic feature, instead its existenc e predates the divergence of archaea and eukaryotes.