Substrate requirements for a novel archaeal endonuclease that cleaves within the 5' external transcribed spacer of Sulfolobus acidocaldarius precursor rRNA.
During ribosome biogenesis in the hyperthermophilic archaeon Sulfolobus acidocaldarius, at least three separate precursor endonucleolytic cleavages occur within the 144-nucleotide-long 5' external transcribed spacer (5' ETS) region of the rRNA operon primary transcript. The 5' ETS sequence contains three regions of very stable helical structure. One cleavage (5' to position -98) is in the single-stranded region between the 5' and the central helical domains; a second cleavage (5' to position -31) is in the single-stranded region between the central and the 3' helical domains; and a third cleavage is at the 5' ETS-16S junction (5' to position +1). The three sites share a common consensus sequence around the position of cleavage. We have used an in vitro pre-RNA processing assay to define some of the sequence and structural recognition elements necessary for the two precursor cleavages 5' to positions -98 and -31. Surprisingly, none of the three predominant helical domains are required for recognition or targeting of the cleavages, although their removal reduces the rate of cleavage site utilization. We show that the sequence AAG downward arrow (CA)UU encompassing each site contains at least some of the essential features for recognition and efficient targeting of the cleavages. Cleavage depends on the presence of a purine 5' and a uracil two nucleotides 3' to the scissile phosphodiester bond. Mutations to other bases at these critical positions are either not cleaved or cleaved very poorly. Finally, on the basis of intermediates that are produced during a processing reaction, we can conclude that the cleavages at positions 98 and 31 are not ordered in vitro.
Russell
A G
AG
Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada.
Ebhardt
H
H
Dennis
P P
PP
D001105:Archaea / Q000201:enzymology*; D019843:Archaeal Proteins / Q000378:metabolism*; D001483:Base Sequence; D016384:Consensus Sequence; D004722:Endoribonucleases / Q000378:metabolism*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012322:RNA Precursors / Q000378:metabolism*; D012323:RNA Processing, Post-Transcriptional; D019642:RNA, Archaeal / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism; D017384:Sequence Deletion; D013379:Substrate Specificity; D016944:Sulfolobus / Q000235:genetics*
D019843:Archaeal Proteins; D012322:RNA Precursors; D019642:RNA, Archaeal; D004722:Endoribonucleases