Site selection by Xenopus laevis RNAase P

Carrara, G.; Calandra, Patrizia; Fruscoloni, Paolo; Doria, Margherita; Tocchini‐Valentini, Glauco P.

doi:10.1016/0092-8674(89)90400-5

Cited by 48 publications

(31 citation statements)

References 24 publications

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“…2 (Fig. 1A), which is cleaved by RNase P one nucleotide downstream of the wild-type cleavage site (8). CPA permits one to determine which of the possible circularly permuted linear precursor molecules can be cut by RNase P. In CPA a unique position in the circular sequence is labeled with 32p; in our experiments this was the phosphate on the 5' side of nucleotide G-1.…”

Section: Resultsmentioning

confidence: 99%

“…The identification of the 5'-terminal oligonucleotides produced by RNase P cleavage was carried out on a 20% polyacrylamide gel. The leader sequence comigrates with the 9-base oligonucleotide produced by digestion of the wild-type precursor with RNase Pl (an endonuclease that forms nucleoside 5'-monophosphates), indicating the presence of a 3'-OH terminus on the product (8).…”

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confidence: 99%

“…The enzyme requires magnesium ions, and the end groups produced during the cleavage reaction are 5'-P and 3'-OH (8).…”

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confidence: 99%

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Two helices plus a linker: a small model substrate for eukaryotic RNase P.

Carrara

Calandra

Fruscoloni

et al. 1995

Proc. Natl. Acad. Sci. U.S.A.

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Using precursor tRNA molecules to study RNA-protein interactions, we have identified an RNA motif recognized by eukaryotic RNase P (EC 3.1.26.5). Analysis of circularly permuted precursors indicates that interruptions in the sugar-phosphate backbone are not tolerated in the acceptor stem, in the T stem-loop, or between residues A-9 and G-10. Prokaryotic RNase P will function with a minihelix consisting of the acceptor stem connected directly to the T stem-loop. Eukaryotic RNase P cannot use such a minimal substrate unless a linker sequence is added in the gap where the D stem and anticodon stem-loop were deleted.Molecules of tRNA are synthesized as precursors, which, in turn, are converted to mature tRNA by a series of enzymatic reactions (1). The endonuclease RNase P (EC 3.1.26.5) is responsible for the generation of the 5' termini of mature tRNA molecules from their precursors (2). A variety of evidence indicates that, in both prokaryotes and eukaryotes, a single enzyme can cleave several precursors. Therefore, the enzyme must be dealing directly or indirectly with features that are shared by all the precursors. The precursors probably present a tRNA-like tertiary structure in the mature domain of the molecule, even when an intervening sequence is present. Calculations of free-energy minima (3) and the use of chemical and enzymatic structure-specific probes (4) suggest that all of the precursor tRNAs (pre-tRNAs) have a common tertiary structure (5, 6). In this structure, the tRNA portion of the precursor maintains the L-shaped conformation that is stabilized by the interaction between the D and TTC loops.We previously described a highly purified preparation of RNase P from Xenopus laevis oocyte nuclei (germinal vesicles) (7). The enzyme requires magnesium ions, and the end groups produced during the cleavage reaction are 5'-P and 3'-OH (8).How is cleavage site specificity determined? Because the lengths of the leaders and their sequences are not, in general, conserved among different pre-tRNAs (9, 10), the main cleavage site determinants must be in the mature domain (11,12).To obtain information on the features of the pre-tRNAs recognized by Escherichia coli RNase P, McClain et al. (13) produced mutant precursors that lack specific domains of the normal tRNA sequence. The smallest tRNA precursor that was cleaved efficiently retained only the helical segment of the amino acid acceptor stem and the T stem-loop. The implication of these results is that the reduced substrate contains determinants for precursor recognition and cleavage.Can a smaller substrate be defined for the eukaryotic enzyme?MATERIALS AND METHODS pre-tRNA Circularization. The yeast pre-tRNAPhe gene and the variant with an A-U base pair insertion in the pre-tRNA acceptor stem (AUV acc.s. pre-tRNAPhe), both of which were placed under the control of the bacteriophage T7 promoter, were assembled from a set of 10 synthetic oligodeoxynucleotides (14). The T7 promoter/pre-tRNAPhe gene constructs were inserted into the Pst I/BamHI site of the v...

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Section: Resultsmentioning

confidence: 99%

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confidence: 99%

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Two helices plus a linker: a small model substrate for eukaryotic RNase P.

Carrara

Calandra

Fruscoloni

et al. 1995

Proc. Natl. Acad. Sci. U.S.A.

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“…Deletion, replacement and point mutagenesis have indicated no primary sequence and a distance requirement for RNA processing by RNase MRP [4]. Substrate RNA folding may be effected by several of these mutations and therefore -similar to RNase P -the particular folding of the substrate RNA into a certain structure may be the predominant determinant for cleavage site selection by RNase MRP [5,38,40,43]. Experimental analysis of wildtype and mutant D-loop mtRNA folding and its influence on RNase MRP cleavage site selection will [19]; mouse La9 cell MRP RNA [7]; Xenopus luevis MRP RNA [20].…”

Section: Cellular Relation Of Rnase P and Rnase Mrpmentioning

confidence: 99%

“…The most important domains for the recognition of pre-tRNAs by RNase P are contained in the acceptor stem and T arm, with the acceptor stem being a critical element for the selection of the precise cleavage site [34][35][36][37][38][39][40][41]. Eucaryal RNase P appears less flexible than bacterial RNase P and thus may require different features for pre-tRNA processing [34,39]: while bacterial RNase P (or Ml RNA, the catalytic RNA component of the E. coli RNase P [5,34]) cleaves minimal model substrates by means of a simple 'external guide sequence' (EGS, contained in the acceptor stem and T 2 stem) [34-361 eucaryal RNase P enzymes do not cleave these minimal model substrates [34,36,39,41].…”

Section: Cellular Relation Of Rnase P and Rnase Mrpmentioning

confidence: 99%

RNase MRP/RNase P: a structure‐function relation conserved in evolution?

Karwan

1993

FEBS Letters

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RNase P and RNase MRP are related ribonucleoproteins. RNase MRP processes mitochondrial precursor-(primer) RNAs, whereas RNase P cleaves precursor-tRNAs to produce their mature S-ends. Both RNase P and RNase MRP are associated with the Th/To ribonucleoprotein suggesting possible interrelated pathways and/or functions. All known RNase P and RNase MRP RNAs contain conserved structural elements possibly involved in catalysis/substrate binding, but these elements do not predict all cellular functions of the RNPs.

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