Separate pathways for excision and processing of 16S and 23S rRNA from the primary rRNA operon transcript from the hyperthermophilic archaebacterium Sulfolobus acidocaldarius: similarities to eukaryotic rRNA processing.

Abstract

In the hyperthermophilic archaebacterium Sulfolobus acidocaldarius, the mature 16S and 23S rRNA are generated by processing of a 5000-nucleotide transcript. Analysis of intermediates that accumulate in vivo indicates that the transcript contains 11 separate processing sites. The processing and maturation of 23S rRNA appears to follow the typical archaebacterial pathway, utilizing a bulge-helix-bulge motif within the 23S processing helix as the substrate for an excision endonuclease. The precursor 23S rRNA that is released is trimmed at its 5' and 3' ends to generate the mature 23S rRNA found in 50S ribosomal subunits. The pathway for processing and maturation of 16S rRNA is distinctive and does not use the bulge-helix-bulge motif in the 16S processing stem. Instead, the transcript is cleaved at several novel positions in the 5' leader and in the 3' intercistronic sequence. The excised precursor 16S is trimmed at the 5' end but an extra 60 nucleotides of what is normally spacer sequence is retained at the 3' end. The elongated 16S rRNA is present in active 30S subunits. An in vitro processing system for the 16S rRNA has been established. The RNA substrate containing the entire 144-nucleotide 5' leader and the first 72 nucleotides of 16S sequence is cleaved at the same positions observed in vivo by an endonuclease activity present in cell extract. These results demonstrate (i) that the 16S processing helix is neither utilized nor required for leader processing, and (ii) that complete maturation to the 5' end of 16S rRNA can occur in the absence of concomitant ribosome assembly and in the absence of all but the first 72 nucleotides of the 16S rRNA sequence. The endonuclease activity responsible for cleavage of the 5' leader substrate is sensitive to nuclease digestion, suggesting that it contains an essential RNA component. The cleavage sites appear to be located within regions of irregular secondary structure and have a consensus sequence of GAUUCC.