In vitro processing of the 16S rRNA of the thermophilic archaeon Sulfolobus solfataricus

Abstract

In this paper we have analyzed the processing in vitro of the 16S rRNA of the thermophilic archaeon Sulfolobus solfataricus, using pre-rRNA substrates transcribed in vitro and different protein preparations as the source of processing enzymes. We show that the 5' external transcribed spacer of the S. solfataricus pre-rRNA transcript contains a target site for a specific endonuclease, which recognizes a conserved sequence also existing in the early A0 and 0 processing sites of Saccharomyces cerevisiae and vertebrates. This site is present in other members of the kingdom Crenarchaeota but apparently not in the Euryarchaeota. Furthermore, S. solfataricus pre-16S RNA is processed within the double-helical stem formed by the inverted repeats flanking the 16S RNA sequence, in correspondence with a bulge-helix-bulge motif. The endonuclease responsible for this cleavage is present in both the Crenarchaeota and the Euryarchaeota. The processing pattern remained the same when the substrate was a 30S ribonucleoprotein particle instead of the naked RNA. Maturation of either the 5' or the 3' end of the 16S RNA molecule was not observed, suggesting either that maturation requires conditions not easily reproducible in vitro or that the responsible endonucleases are scarcely represented in cell extracts.

FIG. 1

In vitro processing of the short pre-rRNA by different protein preparations and processing of a minimal transcript. (A) Primer extension analysis of the processing cuts introduced in the short transcript by the following protein preparations: chaperonin (Ch), the postribosomal S-100 fraction (S100), and the RW fraction. The short transcript, which included the entire 5′ETS and about 100 nt of the 16S rRNA sequence, is illustrated on the right. The sequence and secondary structure of the 5′ETS are shown in full, while the remainder of the molecule is schematized as a box (16S, 100 nt). The primary processing site (site 0) and the location of the mature 5′ end of the 16S RNA are indicated. (B) In vitro processing patterns of uniformly labeled RNA molecules. Lanes 1 and 2, short transcript incubated with and without chaperonin, respectively; lanes 3 and 4, minimal transcript incubated in the same way. The structure of the minimal transcript (about 100 nt) is illustrated on the right.

FIG. 2

In vitro processing of the complete 16S pre-rRNA. The structure of the long transcript is schematized on the right. The sequences and secondary structures of the 5′ETS and of the ITS are shown in full, while the 16S sequence is represented schematically as a loop on top of the processing stem. The locations of the processing sites (site 0, site 1, and site 1′) and that of the 5′ terminus of the 16S rRNA are indicated. The left and top panels show the positions of the processing cuts as revealed by primer extension experiments. Ch, chaperonin.

FIG. 3

Site 0 is located in a single-stranded region, and cleavage is sequence specific. (Left panel) Primer extension analysis performed on an unmodified short transcript (lane −) and on the same transcript modified with CMCT (lane +). The main stop signals corresponding to modified uracil residues are indicated with arrows; the sequence at site 0 is evidenced. (Top middle panel) Alignment of the sequences around site 0 in several archaea and around sites A0 and 0 in yeast and mouse. The positions of the processing cuts, when known, are marked with arrows. The conserved CUU motif is underlined. (Bottom middle panel) Analysis by site-directed mutagenesis of the sequence determinants essential for processing at site 0. The nucleotides modified in each experiment are underlined; the efficiency of processing was assayed by incubating a uniformly labeled minimal transcript with the purified chaperonin. +, complete cleavage; +/−, partial cleavage; −, no cleavage. (Right panel) Structure of the short transcript. The uracil residues modified by CMCT are indicated with arrows.

FIG. 4

Cleavage in the processing stem is structure specific. (Left panel) Sequence and predicted structure of the wild-type and mutated processing stems. The modified nucleotides (GG to CU) are boxed. (Right panels) Primer extension analysis of processing at sites 0 and 1 (top) and 1′ (bottom) by the S-100 fraction in the wild-type (wt) and mutated (mut 1) long transcripts.

FIG. 5

Processing on ribonucleoprotein particles. (Top panel) Primer extension analysis of the processing cuts introduced by either the chaperonin (Ch) or the S-100 proteins on a long pre-rRNA, in the absence and in the presence of the total small-subunit proteins (TP30). (Bottom panel) Density gradient analysis showing that the TP30 and the long pre-rRNA assemble to form a 30S particle when incubated at 80°C under the conditions described in Materials and Methods. The gradients, 10 to 30% sucrose in reconstitution buffer, were run for 4 h at 38,000 rpm in a Beckman SW41 rotor.

FIG. 6

Processing of the S. solfataricus pre-16S RNA by heterologous enzymes. The results of primer extension analysis of in vitro cleavage at sites 0 and 1 of a long transcript in the presence of the S-100 fractions from S. solfataricus (Sso), D. mobilis (Dmo), and T. celer (Tce) are shown.