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Nucleotide Sequence Surrounding a Ribonuclease III Processing Site In Proc. Nati. Acad. Sci. USA Vol. 74, No. 3, pp. 984-988, March 1977 Biochemistry Nucleotide sequence surrounding a ribonuclease III processing site in bacteriophage T7 RNA (intercistronic region/polycistronic mRNA precursor/hairpin structure/endoribonuclease III) MARTIN ROSENBERG* AND RICHARD A. KRAMERt f * Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20014; and t Department of Biochemistry, Stanford University School of Medicine, Stanford, California 94305 Communicated by Charles Yanofsky, January 3, 1977 ABSTRACT We have determined a nucleotide sequence of the 5' end of the gene 0.7 mRNAs, (ii) some fragments that 87 residues surrounding a ribonuclease III (endoribonuclease contain the RNase III cleavage site are not recognized and III; EC 3.1.4.24) processing site in the bacteriophage 17 inter- cistronic region between early genes 0.3 and 0.7. The structural cleaved by the enzyme; and (iii) the 3'-terminal oligoadenylate requirements necessary for proper recognition and cleavage by sequences found on the ends of the in vdvo T7 early mRNAs (11) RNase III are discussed. In addition, other structural features are not encoded by the genome, but presumably represent a characteristic of this intercistronic boundary are described. nontemplate-dependent post-transcriptional modification. Here, we report the complete nucleotide sequence of the gene When bacteriophage T7 infects Escherichia coli, the host RNA 0.3-0.7 intercistronic region of T7 and propose a specific role polymerase (RNA nucleotidyltransferase, EC 2.7.7.6) tran- for RNA secondary structure in substrate recognition and action scribes only the early region of the phage genome (i.e., leftmost of RNase III. 20%, Fig. 1) (2-4). Transcription initiates at three closely spaced sites near the left end of the DNA and terminates predomi- nantly at a single site at the distal end of the early region (4-6). MATERIALS AND METHODS The resulting transcript is a high-molecular-weight (2.2 X 106), polycistronic mRNA precursor which is subsequently cleaved Preparation of Polycistronic T7 Early RNA. RNA was at specific sites into individual monocistronic mRNAs by ri- synthesized in vitro from T7 DNA as previously described (12). bonuclease III (RNase III; endoribonuclease III; EC 3.1.4.24) Fragments of this transcript that spanned the gene 0.3-0.7 in- a host-specified endonuclease (7, 8). RNase III is known to tercistronic boundary were isolated by a two-step hybridization function in E. coli as a "processing" enzyme involved in the procedure (outlined in Fig. 1) using DNA obtained from ap- normal maturation of the primary ribosomal RNA transcription propriate deletion mutants of T7. The procedure and prelim- products (8, 9). inary characterization of the RNA fragments obtained have The individual T7 early RNAs can be either isolated directly been described in detail elsewhere (1). Deletion phage strains from T7-infected cells or synthesized in vitro by transcription C116, C114, C74, and H3 (17) were provided by F. W. Stu- of T7 DNA with E. coli RNA polymerase and digestion of the dier. RNA products with purified RNase III (3-5, 10). We have Fingerprint Analysis. Digests of the RNA fragments were previously examined sequences at the 5' and 3' termini of the prepared with T1 and pancreatic ribonucleases and analyzed early mRNA species obtained in both ways (11-13). Our results by standard RNA sequencing techniques (18, 19). The proce- indicated that the in vivo mRNAs are the direct products of dures and the results of these analyses have been previously RNase III cleavage. In addition, we found that the processed published (1). RNAs have identical 3'-terminal sequences (C-C-U-U-U-A- Partial Enzymatic Digestion of the RNA Fragments: UOH) as well as identical 5'-terminal sequences (pG-A-U). This Separation and Characterization of Large Oligonucleotide conserved structure at the various T7 cleavage sites suggested Products. Limited enzymatic digestion of the RNA fragments that sequence specificity might be important to the processing with either T, or pancreatic RNase was carried out in 0.01 M event. Moreover, the fact that RNase III is known to exhibit a Tris-HCl (pH 7.5), 0.02 M MgCl2, at 40 for 20-40 min; en- general specificity for double-stranded RNA (although it de- zyme-to-substrate ratios varied from 1:250 to 1:1000. The grades this RNA rather non-specifically) (14-16) suggests that products were then fractionated by standard two-dimensional single-stranded RNAs that serve as natural substrates for RNase techniques; electrophoresis on Cellogel (Kalex) in 8.0 M urea III might contain specific double-stranded regions that are at pH 3.5 in the first dimension and homochromatography on recognized by the enzyme. thin-layer plates of DEAE-cellulose (Analtech; 9:1, cellulose: To elucidate further the action of RNase III in processing DEAE-cellulose; 40 X 20 cm) in the second. Homochromato- natural substrates, we have isolated and characterized several graphy solutions (18, 19) were used which effectively separated overlapping RNA fragments that span the intercistronic region oligomers up to 45 residues in chain length. Alternatively, between the T7 early genes 0.3 and 0.7 (1). Preliminary analysis partial digestion products were separated by electrophoresis of these fragments indicated that: (i) RNase III produces a single on 15% polyacrylamide slab gels containing 8.0 M urea using endonucleolytic break between the 3' end of the gene 0.3 and Tris-borate, pH 8.3, as running buffer (20); bands were located autoradiographically and the samples were eluted as previously Abbreviations: RNase, ribonuclease; 0.3 mRNA, 0.7 mRNA, 1.0 described mRNA, and 1.1 mRNA, the messenger RNAs of bacteriophage T7 for (11). the protein products of genes 0.3, 0.7, 1.0, and 1.1, respectively; V, All partial products were further characterized by complete deletion mutant. digestion with the appropriate enzymes and fractionation in t Present address: Laboratory of Biochemistry, National Cancer In- either one or two dimensions by standard electrophoretic stitute, National Institutes of Health, Bethesda, Md. 20014. and/or chromatographic techniques (18, 19, 21). 984 Downloaded by guest on September 26, 2021 Biochemistry: Rosenberg and Kramer Proc. Natl. Acad. Sci. USA 74 (1977) 985 PE G 0.3 G 0.7 G 1.0 G 1.1 G 1.3 TE T7 DNA 7,, % 0 1.0 it 3.5 8.0 - 1. - A B 0 I ICUIG 1,) UG EARLY 5'l / i I 3 G~)AG C:) RNA __ _- 0 C~DAWuG '-- ICA)G OAAG C) HYBRIDIZATION t19 0 D)2 01 02 °3 I OF RNA TO VC1 16 '- -018 /- 11-1 f, n G .3 U0., 051 oOQQ004 5 6 0° 7C116 3.3 5.8 8.0 C)I9l 14 | UNHYBRIDIZED 1 09 '010 212 RNA L o I 0 8 Q6 00 0 (7 (3 13 14 15 |() HYBRIDIZATION OF RNA TO 0 05 I0 *(a) VC74 or (b)VC1 14 or (c) V H3 4or 0 °16 04 0 3.5 8.0 0 Q3a xo 07 17_ a) 7C74 03b 03: 0 cc 0 Or 3.7 7.5 0 02 b)VC114 pH 3.5 - 01~ pH 3.5 - Or 3.9 7.6 c) V H3 FIG. 2. Composite sketches of the two-dimensional fingerprints | RNA Fragments obtained from Ti (A) and pancreatic (B) RNase digestion ofthe iso- Obtained lated fragments. The assignment of particular oligonucleotides to a) C74 33 3.5 specific regions ofthe template is indicated in Table 1. For comparison bi C1 14 33 _ 37 with the autoradiographs of these fingerprints, see Kramer and C) H3 33 39 Rosenberg (1). FIG. 1. Diagrammatic outline of the procedure used to isolate RNA fragments containing an RNase III cleavage site from the gene positioned in this manner. The data are summarized in Table 0.3-0.7 intercistronic boundary of the T7 early RNA precursor. RNA 1 and Fig. 2. is synthesized in vitro from wild-type T7 DNA and hybridized on Determination of the Complete Sequence; Oligonucleo- filters to the DNA from T7 deletion mutant C116. Under appropriate tide Overlaps. Partial and pancreatic RNase digestions were hybridization conditions (1) the region of the transcript that corre- T, sponds to the deleted segment ofDNA does not hybridize to the filter carried out on the three sets of RNA fragments and the products and remains in the supernatant portion of the hybridization mixture were fractionated and analyzed as described in Materials and (step 1: unhybridized RNA). This RNA is then hybridized to DNA Methods. The results (Fig. 3) are consistent with the deletion from one of the T7 deletion mutants C74, C114, or H3 (step 2). Only mapping data presented above and allow deduction of an un- RNA derived from the leftmost segment of the C116 deletion will ambiguous sequence of 87 residues. Partial digests were also hybridize to these deletions. The filter-bound hybrids are trimmed performed on each of the three sets of RNA fragments after with ribonuclease (to remove unhybridized "tails") and subsequently eluted from the DNA. The RNA fragments obtained span the gene initial treatment with purified RNase III (12). Analyses of the 0.3-0.7 boundary and contain a single RNase III cleavage site. All products confirmed the sequence shown in Fig. 3 and support distances given are in T7 map units: 1 map unit = 1% of T7 genome our earlier conclusions that (i) RNase III produces a single en- = 380 base pairs. PE, region of initiation for T7 early transcription; donucleolytic break in this region of the T7 mRNA and (ii) TE, site of termination of T7 early transcription; i, an RNase III although all of the RNA fragments contain the site of RNase cleavage site; v, deletion mutant of T7.
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