Processing in vitro of precursor tRNA^Tyr with 6XHis-Rpp 14. Assay in vitro of 3′ processing of precursor tRNA^Tyr containing 5′ and 3′ extra sequences (denoted as 5′-Tyr-3′) by 6XHis-Rpp 14. Aliquots of each immunoprecipitate and substrate concentration used in each reaction are given in the legend of Fig. 1. The reaction products were separated on a 12% sequencing gel. Lanes 1–8, internally ³²P-labeled 5′-Tyr-3′ as substrates; lanes 1 and 2, substrates incubated for 0 or 45 min as controls; lanes 3 and 4, time course with immunoprecipitated 6XHis-Rpp 20 for 20 or 45 min; lanes 5 and 6, immunoprecipitated 6XHis-Rpp 14 for 20 or 45 min; lanes 7 and 8, immunoprecipitated human RNase P from HeLa cells crude extracts by anti-Rpp 29 polyclonal antibody for 20 or 45 min. Several faint bands appear in the lane below Tyr-3′, an indication of processing at the 3′ terminus. Lanes 9–16, 5′-³²P-labeled 5′-Tyr-3′ as substrates; lanes 9 and 10, substrate incubation for 0 or 45 min as controls; lanes 11 and 12, time course with immunoprecipitated 6XHis-Rpp 20 for 20 or 45 min; lanes 13 and 14, with immunoprecipitated 6XHis-Rpp 14 for 20 or 45 min; lanes 15 and 16, with immunoprecipitated human RNase P from HeLa cells crude extracts by anti-Rpp 29 polyclonal antibody for 20 or 45 min. Major products are indicated on both sides of the figure. Tyr-3′ was the precursor of tRNA^Tyr, of which 5′ leader sequence was removed; 5′-Tyr was a processed precursor of tRNA^Tyr from which the trailing 3′ sequence was removed; 5′ is the 5′ leader sequence.

Requirements of the reactions governed by recombinant Rpp 14 and OIP2. Assays in vitro were performed with 6XHis-Rpp 14 or 6XHis-OIP2 immunoprecipitated by anti-polyhistidine monoclonal antibody. Aliquots as defined earlier, as was substrate concentration (Fig. 1). Time of incubation was 10 min at 37°C. (A) Effect of Mg²⁺ on 3′ processing of internally labeled (with [α-³²P]UTP) SupS1-3′ by 6XHis-Rpp 14 and 6XHis-OIP2. Lane 1, substrate; lane 2, substrate incubation in the presence of 5 mM MgCl₂ as control; lanes 3–9, 3′ end processing with 6XHis-OIP2 in the presence of 0, 1, 2.5, 5, 10, 20, and 40 mM MgCl₂, respectively; lanes 10–16, 3′ end processing with 6XHis-Rpp 14 in the presence of 0, 1, 2.5, 5, 10, 20, and 40 mM MgCl_2, respectively. (B) Phosphate-dependent processing of SupS1-3′ by 6XHis-Rpp 14 and 6XHis-OIP2. Lane 1, substrate; lane 2, substrate incubation in the presence of 15 mM phosphate as control; lanes 3–11, 3′ end processing with 6XHis-Rpp 14 in the presence of 0, 2.5, 5, 10, 20, 40, 80, 160, and 320 mM of phosphate, respectively; lanes 12–20, 3′ end processing with 6XHis-OIP2 in the presence of 0, 2.5, 5, 10, 20, 40, 80, 160, and 320 mM phosphate, respectively. The reaction products were separated on an 8% polyacrylamide gel. Mononucleotides are not shown on the gel.

5′ and 3′ processing of precursor tRNA by human RNase P and its associated exonucleolytic components: HeLa cell crude extracts subject to immunoprecipitation assay were precleaned with preimmune sera coupled to protein A-Sepharose beads. The immunoprecipitates were obtained by mixing the precleaned HeLa cell crude extracts with beads coupled to the preimmune rabbit sera as control, and with beads coupled to anti-OIP2 or anti-Rpp 29 polyclonal antibodies. The immunoprecipitate aliquots above (defined in Materials and Methods and amount of substrate used as in Fig. 1) were assayed for tRNA processing (times indicated in the figure) by using internally ³²P-labeled precursor tRNA^Tyr containing 5′ and 3′ extra sequences (denoted as 5′-Tyr-3′) or internally ³²P-labeled precursor tRNA^Tyr containing only 5′ extra sequences (denoted as 5′-Tyr). (A) Lanes 1–12, internally ³²P-labeled 5′-Tyr-3′ as substrates. Lane 1, input substrate; lane 2, digestion with highly purified human RNase P (the positions of processed products pTyr-3′ and 5′ leader sequence were marked on the right side of the figure); lane 3, substrate incubation for 30 min; lanes 4–6, immunoprecipitates by preimmune sera; lanes 7–9, immunoprecipitates by anti-OIP2 antibody; lanes 10–12, immunoprecipitates by anti-Rpp 29 polyclonal antibody; lanes 13–19, internally ³²P-labeled 5′-Tyr as substrates; lanes 13 and 15, substrate input; lane 14, digestion with purified RNase P for 10 min; lane 16, substrate incubation for 10 min; lane 17, digestion with immunoprecipitated human RNase P by anti-Rpp 29 polyclonal antibody for 10 min; lane 18, digestion with immunoprecipitated OIP2 by anti-OIP2 polyclonal antibody for 10 min; lane 19, digestion with immunoprecipitates by preimmune sera for 10 min. Major products are indicated on the left side of the figure. mTyr is mature tRNA; 5′ is the 5′ leader sequence. (B) Identification of products with internally ³²P-labeled precursor SupS1 that contains only 5′ leader sequences (denoted as 5′-SupS1) or internally ³²P-labeled precursor SupS1 containing only 3′ extra sequences (prepared by digestion of 5′-SupS1-3′ with human RNase P, denoted as SupS1-3′). After the reaction was finished, the processed products were analyzed in a 5% sequencing gel. Lane 1, 5′-SupS1 incubation; lane 2, 5′-SupS1 incubation with RNase P (produces a mature SupS1 of 82 nucleotides); lane 3, ladder made from mixture of four sequencing reactions of a DNA sample; lanes 4–6, SupS1-3′ incubation with immunoprecipitated 6XHis-OIP2, 6XHis-Rpp 14, and 6XHis-Rpp 20 for 30 min, respectively; lanes 7–9, 11, 12, 5′-SupS1-3′ incubation alone, with RNase P, immunoprecipitates by preimmune sera, immunoprecipitates by anti-Rpp 29 polyclonal antibody and HeLa cell extracts for 30 min, respectively; lanes 14 and 15, 5′-SupS1 incubation with buffer or with RNase P. The tRNAs and their size are labeled on both sides of the figure. The number 82 shows the size of mature SupS1 and is counted as a standard along the sequencing ladder. Therefore, number 88 is the size of partially 3′ end-processed products, indicating that the products have six extra nucleotides after the mature 3′ end.

Recombinant Rpp 14 and OIP2 exhibit 3′→5′ exoribonuclease activity in vitro. Assays in vitro were performed with 6XHis-Rpp 14, 6XHis-Rpp 20, or 6XHis-OIP2 immunoprecipitated by antipolyhistidine monoclonal antibody (Sigma), and the reaction products were analyzed by PAGE (see Materials and Methods). (A) 6XHis-Rpp 14, 6XHis-Rpp 20, or 6XHis-OIP2 were expressed in E. coli and then immunoprecipitated with antipolyhistidine monoclonal antibody bound to protein A-Sepharose CL-4B beads. The measurement of an aliquot of each immunoprecipitate is defined in Materials and Methods. One aliquot of each sample was boiled in 1× SDS-polyacrylamide gel-loading buffer, separated on a SDS-12% polyacrylamide gel, and visualized by Coomassie brilliant blue staining. Lane 1, anti-polyhistidine antibody bound to protein A-Sepharose CL-4B bead; lane 2, 6XHis-Rpp 20 precipitated with antipolyhistidine antibody bound to protein A-Sepharose CL-4B bead (the arrow indicates 6XHis-Rpp 20); lane 3, 6XHis-Rpp 14 precipitated with anti-polyhistidine antibody bound to protein A-Sepharose CL-4B bead (the arrow indicates 6XHis-Rpp 14); lane 4, 6XHis-OIP2 with anti-polyhistidine antibody bound to protein A-Sepharose CL-4B bead (the arrow indicates 6XHis-OIP2). For reference purposes, the approximate molecular weight of the OIP2 protein is 32–36 kDa and that of ribonuclease PH is 22–25 kDa. (B) Assay of 6XHis-Rpp 14, 6XHis-Rpp 20, and 6XHis-OIP2 with 5′-³²P-labeled AEF RNA. One aliquot of each immunoprecipitate was used for each assay shown in B–D. Substrate was approximately 0.05 nmol per reaction. Reaction products were resolved in a 12% polyacrylamide gel. Time course with substrate alone (lanes 1–4), 6XHis-Rpp 20 (lanes 5–7), 6XHis-Rpp 14 (lanes 8–10), or 6XHis-OIP2 (lanes 11–13). Time (minutes) indicated in the figure. (C) Time course of processing of 5′-³²P-labeled AEF RNA by 6XHis-Rpp 14 in the absence of exogenously added 15 mM Na₂HPO₄ (pH 8.0) or 5 mM MgCl₂. Lane 1, substrate; lanes 2–5, 6XHis-Rpp 20 in the presence of Na₂HPO₄ and MgCl₂; lanes 6–17, 6XHis-Rpp 14 in presence of both Na₂HPO₄ and MgCl₂(lanes 6–9), only MgCl₂ (lanes 10–13) or only Na₂HPO₄ (lanes 14–17), respectively. (D) Time course of processing of 5′-³²P-labeled AEF RNA by 6XHis-OIP2 in absence of exogenously added 15 mM Na₂HPO₄ (pH 8.0) or 5 mM MgCl₂. Lane 1, substrate; lanes 2–5, substrate incubated in the presence of Na₂HPO₄ and MgCl₂; lanes 6–17, 6XHis-OIP2 in presence of both phosphate and MgCl₂ (lanes 6–9), only MgCl₂ (10–13), or only phosphate (lanes 14–17), respectively.

Thin-layer chromatographic analysis of the products released by the 6XHis-Rpp 20, 6XHis-Rpp 14, 6XHis-OIP2, and human RNase P-associated exoribonuclease activity. Aliquots for each immunoprecipitate and substrate concentration are given in the legend to Fig. 1. The reactions were performed as described in Materials and Methods with 5′-SupS1-3′ transcribed in the presence of [α-³²P]UTP. The standards are UTP, UDP, and UMP. The reaction products were mixed with the standards and were resolved by chromatography on polyethyleneimine-cellulose thin-layer plates. Lanes 1–4, 5′-SupS1-3′ incubated for 10 min at 37°C with 6XHis-Rpp 20, 6XHis-Rpp 14, 6XHis-OIP2, and immunoprecipitated human RNase P by anti-Rpp 29 polyclonal antibody (anti-p29), respectively. The standards were visualized with UV light and their positions are shown at the left side of the figure. The sample application points are indicated by an arrowhead, the direction of chromatography was from bottom to top.

Exoribonuclease assay of immunoprecipitates from HeLa cells by anti-human RNase P subunits (Rpp 14, Rpp 20, and Rpp 29) polyclonal antibodies. HeLa cell crude extracts subject to immunoprecipitation assay were first cleaned with preimmune sera coupled to protein A-Sepharose beads. The immunoprecipitates were obtained by mixing the cleaned HeLa cell crude extracts with beads coupled to the preimmune rabbit sera as control, and with beads coupled to anti-Rpp 14, anti-Rpp 20, or anti-Rpp 29 as polyclonal antibody. The immunoprecipitates above, aliquots assayed as described in Materials and Methods, were assayed for exoribonuclease activity by using 5′-³²P-labeled AEF RNA substrates. Lanes 1–3, substrate incubation; lanes 4–6, immunoprecipitates by preimmune sera; lanes 7–9, immunoprecipitates by anti-Rpp 20 polyclonal antibody; lanes 10–12, immunoprecipitates by anti-Rpp 14 polyclonal antibody; lanes 13–15, immunoprecipitates by anti-Rpp 29 polyclonal antibody.