The nucleotide sequence of a precursor to the glycine- and threonine-specific transfer ribonucleic acids of Escherichia coli.

The primary nucleotide sequence of an Escherichia coli tRNA precursor molecule has been determined. This precursor RNA, specified by the transducing phage lambdah80dglyTsuA36 thrT tyrT, accumulates in a mutant strain temperature-sensitive for RNase P activity. The 170-nucleotide precursor RNA is processed by E. coli extracts to form mature tRNA Gly 2 suA36 and tRNA Thr ACU/C. The sequence of the precursor is pG-U-U-C-C-A-G-G-A-U-G-C-G-G-G-C-A-U-C-G-U-A-U-A-A-U-G-G-C-U-A-U-U-A-C-C-U-C-A-G-C-C-U-N-C-U-A-A-G-C-U-G-A-U-G-A-U-G-C-G-G-G-T-psi-C-G-A-U-U-C-C-C-G-C-U-G-C-C-C-G-C-U-C-C-A-A-G-A-U-G-U-G-C-U-G-A-U-A-U-A-G-C-U-C-A-G-D-D-G-G-D-A-G-A-G-C-G-C-A-C-C-C-U-U-G-G-U-mt6A-A-G-G-G-U-G-A-G-m7G-U-C-G-G-C-A-G-T-psi-C-G-A-A-U-C-U-G-C-C-U-A-U-C-A-G-C-A-C-C-A-C-U-UOH(tRNA sequences are italicized). It contains the entire primary nucleotide sequences of tRNA Gly2 suA36 and tRNA Thr ACU/C, including the common 3'-terminal sequence, CCA. Nineteen additional nucleotides are present, with 10 at the 5' end, 3 at the 3' end, and the remaining 6 in the inter-tRNA spacer region. RNase P cleaves the precursor specifically at the 5' ends of the mature tRNA sequences.

From the Section of Biochemistry and Molecular Biology, Department of Biological Sciences, University of California, Santa Barbara, California 93106 The primary nucleotide sequence of an Escherichia coli tRNA precursor molecule has been determined.
This precursor RNA, specified by the transducing phage Xh80dglyTsuA36 thrT tyrT, accumulates in a mutant strain temperature-sensitive for RNase P activity. The 170.nucleotide precursor RNA is processed by E. coli extracts to form mature tRNA::Y,& and tRNAj$;-,,. The

G-A-U-G-C-G-G-G-T-~-C-G-A-U-U-C-C-C-G-C-U-G-C-C-C-G-C-U-C-C-A-A-G-A-U-G-U-G-C-U-G-A-U-A-U-A-G-C-U-C-A-G-D-D-G-G-D-A-G-A-G-C-G-C-A-C-C-C-U-U-G-G-U-mtGA-A-G-G-G-U-G-A-G-m7G-U-C-G-G-C-A-G-T-*-C-G-A-A-U-C-U-G-C-C-U-A-U-C-A-G-C-A-C-C-A-C-U-CIOH(tRNA sequences are italicized).
It contains the entire primary nucleotide sequences of tRNAl,;$$, and tRNA:i'&,,, including the common 3'.terminal sequence, CCA. Nineteen additional nucleotides are present, with 10 at the 5' end, 3 at the 3' end, and the remaining 6 in the inter-tRNA spacer region. RNase P cleaves the precursor specifically at the 5' ends of the mature tRNA sequences.
Functional stable ribonucleic acid species, such as mature ribosomal RNA or transfer RNA, are not direct transcriptional products but arise from the cleavage or longer precursor molecules. Recently, precursors to Escherichia coli tyrosine tRNA (1,2) and to various bacteriophage T4 specified tRNAs (L5) have been isolated and characterized.
An endonuclease (RNase P) which cleaves the tyrosine tRNA precursor specifically at the 5' end of the tRNA sequence was subsequently purified (6). More recently, E. coli mutants defective for tRNA biosynthesis at various steps of the biosynthetic pathway have been isolated and characterized (7,8). Although our current understanding of tRNA biosynthesis is still incomplete, it is evident that the post-transcriptional processing events include (a) nucleolytic cleavages, (b) nucleotide modifications at specific residues, and, in some cases, (c) enzymatic addition of the terminal -C-C-A,,,, sequences.
While much has been learned about post-transcriptional processing of tRNA precursors recently, the fine structure organization of tRNA genes on the chromosome remains unclear. By tRNA-DNA saturation hybridization, it was estimated that there are 40 to 80 tRNA genes per E. coli chromosome (9)(10)(11)(12). From genetic data on the relatively few tRNA genes that have been mapped, it is apparent that tRNA genes are scattered throughout the genome (13). On the other hand, increasing evidence has accumulated indicating that bacterial tRNA genes might often occur in clusters containing several closely spaced genes. These clusters can contain identical genes, as at glyV (14,15) or sup3 (16); or copies of genes specifying different tRNAs, as at the glyT locus (17). Furthermore, Schedl and Primakoff (7) have observed tRNA precursors containing two or more tRNA sequences in E. coli mutant strain A49 (a temperature-sensitive mutant with respect to RNase P activity), indicating that many tRNAs are synthesized via multi-tRNA precursors. We have investigated the transcription of the glyT thrT tyrT tRNA gene cluster which occurs at 77' on the E. coli chromosome and which specifies the synthesis of tRNA:j:y,&,' tRNA.$;-,,., and tRNA:$& respectively (17). Here we I'eport that this tRNA gene cluster is transcribed, at least in part, as a multi-tRNA precursor. In addition, we have determined the total nucleotide sequence of a di-tRNA precursor to the glycine

Amplification
and Isolation of 32P-labeled tRNA""y-tRNAThr Precursor-Previous studies have shown that a cluster of three tRNA genes (glyT thrT tyrT) lies in the region between argH and thi on the Escherichia coli chromosome. These genes specify the structures of tRNAi$t6, tRNA,T::,,c, and tRNATX:,c, respectively (17, 28). Recently, this segment of bacterial DNA has been incorporated onto a defective hybrid phage designated Xh80dglyTsuA36 thrT tyrT (17). After infection of bacteria by this transducing phage, or induction of lysogenic bacteria harboring this phage, the host cells contain many copies of the transducing phage DNA and therefore the synthesis of the phage-specified tRNAs is selectively amplified. By using this method to prepare relatively large quantities of labeled tRNAs, the total nucleotide sequences of tRNA:$& (29, 30) and tRNA:h,:,,, (31) have been determined. The nucleotide sequence of tRNAzC,2/c is also known (32).
Attempts to detect primary transcriptional products (tRNA precursors) from this gene cluster in phage-infected wild type E. coli cells were unsuccessful, presumably due to the rapid processing reactions which occur in normal cells (2). Schedl and Primakoff (7) have isolated a temperature-sensitive E. coli mutant (strain A49) which accumulates tRNA precursors with a wide range of sizes at the nonpermissive temperature (43"). Strain A49 contains a temperature-sensitive RNase P, an endonuclease responsible for the specific scission of many tRNA precursor molecules at the 5' end of mature tRNA (6). We have therefore investigated the synthesis of tRNAs specified by Xh80dglyTsuA36 thrT tyrT in strain A49 at 43", with the intent of isolating incompletely processed tRNA precursors from the glyT thrT tyrT gene cluster.
A polyacrylamide gel electrophoretic separation of 32Plabeled RNA isolated from A49 and the defective lysogen, A49 (Xh80dglyTsuA36 thrT tyrT), under various conditions is shown in Fig. 1. Labeled RNA isolated from A49 cells infected with Xh80dglyTsuA36 thrT tyrT shows a prominent RNA band (labeled E) when the labeling was carried out at 43" (lane b), but only a trace amount of band E is seen at the permissive temperature (30", lane a). Similarly, when the lysogen, A49 (Xh80dglyTsuA36 thrT tyrT), is induced and labeled at 43", the gel pattern again shows the presence of band E RNA (lane c). Non-lysogenic A49 cells labeled at 43" do not contain band E RNA (lane d). Thus, the accumulation of band E RNA in strain A49 depends not only on elevated temperatures (presumably for thermo-inactivation of RNase P), but also on phage infection or prophage induction.
These characteristics suggest that band E RNA could be a tRNA precursor molecule. 5545 the growth medium when labeling was carried out by prophage induction ("Prophage Induction Method" under "Experimental Procedures").
The preparation migrated as a single band on acidic urea acrylamide gels, and was estimated to be 85 to 90% radiochemically pure by measuring contaminants on fingerprints of T,-RNase digests. Band E RNA has an estimated size of 172 * 5 nucleotides, based on its electrophoretie mobility on polyacrylamide gels using E. coli tRNATy', 5 S RNA, and 6 S RNA as markers (not shown).
In vitro Processing of Band E RNA-Purified 32P-labeled band E RNA was incubated with a cell-free extract prepared from sonicated E. coli strain BF266 (wild type RNase P strain) cells, and the digestion products were then fractionated on a 5% polyacrylamide gel. Two RNA bands (Sl and S2) were resolved on the gel as shown in Fig. 2a. Two-dimensional Sanger fingerprints of complete T, RNase digests of Sl and S2 RNAs (Fig. 3, a and c) were compared with authentic fingerprints of mature tRNAT,hC;,,c (31) and tRNAz:Y,& (29, 30) (Fig. 3, b and d). Although some cross-contamination and incomplete modification of minor bases were observed in the fingerprints of the Sl and S2 RNAs, it was evident that Sl RNA is tRNAzE;,,c and S2 RNA is tRNA$",Z,. This was further confirmed by sequence analysis of the T, RNase oligonucleotides (see below). Thus, band E RNA is apparently a precursor RNA that can be processed to form mature tRNA::Y,& and tRNA&,.
FIG. 2. Autoradiograph of acrylamide gel electrophoresis of in vitro precursor cleavage products. a, digestion of l"*PltRNAG'Y-tRNAT"T precursor with a crude cell extract prepared from Escherichiu coli strain BF266 (wild tvue RNase P strain): lane C. untreated nrecursor: lane S, digestion products of the precursor. Separation was on a 5% acrylamide gel for 19 hours at 300 volts. b, digestion of ["'P ]tRNAG'Y-tRNAThT precursor with RNase P. Separation was on a 12% acrylamide gel for 16 hours at 400 volts.
The 5'-terminal T, fragment (pGp) of both mature glycine and threonine tRNAs was found in nearly molar yield on the fingerprints of Sl and S2 RNAs. However, the 3'-terminal T, oligonucleotides appeared to be somewhat heterogeneous in size. Although in vitro cleavage to generate mature 5' ends occurs by a specific endonucleolytic split, it seems likely that maturation of the 3' ends might occur by exonucleolytic degradation (33).
[32P]tRNAG'y-tRNATh' precursor was digested to completion with T, ribonuclease, and the products were fractionated by two-dimensional ionophoresis as described by Barrel1 (22). The resulting fingerprint is shown in Fig. 4. The sequence of each of the T,-RNase oligonucleotides is given in Table I along with the experimental and theoretical yields. For comparison, Table I also lists the theoretical yields of T oligonucleotides of tRNA"'Y ' SUA36 and tRNAzh,;r,c calculated fromlthe known sequences (30,31) as shown in Fig. 7.
The 5'-terminal fragment of the precursor is pGp(tG), which is the only fragment with 5'-phosphate found in the digests. The same nucleotide is also present in fragment pl7(pG-U-) of the pancreatic RNase digests (see below). Since the mature glycine and threonine tRNA 5' ends are both pG-C-. . ., fragment tG(pGp) does not correspond to the 5'-terminal pGp in either one of the mature tRNAs. Also, it may be seen in Table I that fragments with the sequences, C-U-C-C-A,, or C-A-C-C-AoH, which correspond to the 3' ends of tRNA2cA2,, and tRNAxh,{,,,, are absent in T, digests of the precursor. Thus, the precursor contains different terminal sequences from those of the corresponding mature tRNAs. Every T, oligonucleotide found in digests of the mature tRNAs (except the terminal oligonucleotides discussed above) is also found in T, digests of the precursor. Therefore it was possible to assign a tentative sequence to each of the fragments present in T, digests of both the precursor and the mature tRNAs. These sequences were subsequently verified by alkaline hydrolysis, pancreatic RNase digestion, and, for trinucleotides or longer, by venom phosphodiesterase digestion. after dephosphorylation.
Fragments t19a and t19b were only partially resolved on the T,-RNase fingerprints as was previously observed in tRNA'&,c (31). They were analyzed as a mixture without further separation. Fragments t12a and t12b, C-U-C-A-G-from tRNAxvL,,c and C-A-U-C-G-from tRNA'"'Y 2 S"AS8, were not separated on the fingerprints, and were analyzed separately from the fractionated cleavage products generated in vitro (see below).
Fragments t20u and t24u were each present in variable and fractional molar yield. They were also found consistently in T,-RNase digests of the in uitro generated tRNAs as shown in Fig. 3, a and c. The base composition of oligonucleotide t20u is (U-,2A-)G-, as determined by alkaline hydrolysis. Pancreatic ribonuclease digestion gave the products, U-and A-A-G-, establishing the sequence of t20u as U-A-A-G-. Apparently, this oligonucleotide is the unmodified form of t20 (U-mtGA-A-G-) since their yields were variable and totaled 1 mol. Fragment t24u *has the identical base composition as t24 (C-C-U-N-C-U-A-A-G-) when determined by alkaline hy*drolysis, conditions which convert the modified nucleotide Ap to Ap (30). The pancreatic RNase digests of t24u were analyzed and comp?red with that of t24; the only difference observed was that A-A-G-from t24 was replaced by the unmodified fragment, A-A-G-, in t24u. After complete enzymatic digestion to 3'-mononucleotides followed by paper chromatography in  Systems a and b (22), the mpdified nucleotide Np was found in both t24 and t24u, whereas Ap was only present in t24. Because fragments t24 and t24u have the same base composition and are each present in fractional molar yield totaling 0.8 mol, we conclude that t24u has the sequence C-C-U-N-C-U-A-A-Gand represents an undermodified form of t24 (from tRNA$& see Ref. 30).
Sequence analyses of T,-RNase fragments unique to the precursor are summarized in Table II. Base compositions  and  pancreatic RNase digestions of these precursor-specific T,-RNase oligonucleotides established the sequences of tl, t6, t7, and tll. Further experiments needed to determine the nucleotide sequences of fragments t14, t17, t26, and t27 are described below.
The partial sequence of oligonucleotide t14, as determined by alkaline and pancreatic RNase digestion, is (3C-, U-)A-A-G-.
The loss of a cytosine in the venom phosphodiesterase digestion on 3'-dephosphorylated t14 established that this oligonucleotide is C-(2C-, U-)A-A-G-( Table II). The sequence was further investigated by combined silkworm nuclease and bacterial alkaline phosphatase digestion (26).
The completely dephosphorylated small oligonucleotide products were fractionated on DEAE-paper at pH 3.5, and then sequenced by alkaline hydrolysis and venom phosphodiesterase digestion. Among the products, oligonucleotides with the sequences, C-U-C,, and C-C-A-A-G,,, were found, thus it is concluded that the sequence of t14 is C-U-C-C-A-A-G-.
Oligonucleotide t17 has the composition (2U-, 2C-, A-)G-. The loss of a uridine in the venom phosphodiesterase digestion and the results of the pancreatic RNase digestion establish that this fragment is U-(U-, 2C-)A-G- (Table II). Carbodiimide blocking followed by pancreatic RNase digestion gave the products U-U-C-, C-, and A-G-, which establishes the complete sequence of t17 as U-U-C-C-A-G-.
The sequences of oligonucleotides t26 and t27 could not be determined by analysis of the products from alkali, venom phosphodiesterase, and pancreatic RNase digestion. These two fragments were digested with U, ribonuclease, the products fractionated on DEAE-paper at pH 3.5, and then sequenced by alkaline hydrolysis and venom phosphodiesterase digestion. As shown in Table III Conditions for fingerprinting were as described by Barrel1 (22). Ionophoresis was from right to left on cellulose acetate at pH 3.5, and from top to bottom on DEAE-paper in 7% formic acid. Left, autoradiograph; right, diagram identifying the spots.
complete nucleotide sequences were obtained by partial digestion with venom phosphodiesterase.
Each partial digestion product was further analyzed by U, and pancreatic RNase digestions, and the products from these digestions were in turn analyzed by alkaline hydrolysis and venom phosphodiesterase digestion. The results are summarized in Table IV. Oligonucleotides t26 (C-A-C-C-A-C-U-U,,) and t27 (C-A-C-C-A-C-&,) are found in fractional molar yield and their combined yield is less than 1 mol (0.7 mol). They both contain a free 3'-hydroxyl group and differ only in the presence of a single 3'terminal nucleotide, indicating that they are derived from the heterogeneous 3' ends of the precursor. Pancreatic RNase Digestion Products-A fingerprint of the complete pancreatic RNase digestion products of the precursor is shown in Fig. 5. Combined data from alkaline and T,-RNase digestion and, in some cases, from venom phosphodiesterase digestion established the nucleotide sequences of all of the pancreatic RNase fragments ( Table V). All of the pancreatic RNase fragments of glycine and threonine tRNAs except their 5'.terminal oligonucleotides, pG-C-, are present in the precursor. The additional oligonucleotides derived from precursorspecific regions were identified and their sequences determined as summarized in Table II.

In vitro Precursor
Cleavage with RNase P-Purified [32P]tRNAG1y-tRNAThr precursor was digested with RNase P according to the procedure of Robertson et al. (6) and the cleavage products were fractionated by electrophoresis on 12% polyacrylamide gels. Three products are formed (Fig. 2b): two major bands migrating in the approximate position of mature tRNA (EPl and EP2), and a fast moving component containing a relatively low amount of radioactivity (EP3). Labeled RNAs from these bands were eluted, and their sequences determined as described below. Fragment EP3 was digested with T, or pancreatic RNase and the digests were fractionated by standard two-dimensional fingerprinting techniques. Four products were obtained from each digest, and the sequential analysis of each product was carried out as summarized in Table VI. The nucleotide sequences of all of the oligonucleotides except t32 are established by the data given in Table VI. Oligonucleotide t32 has the structure, U-(U-, SC-)A-G-, which matches the composition of fragment t17 obtained from the complete T, RNase digest of the precursor (Table I); thus it is concluded that t32 has the sequence, U-U-C-C-A-G-.
By overlapping the T, and pancreatic RNase fragments, the sequence of EP3 RNA is established as pG-U-U-C-C-A-G-G-A-U... Two-dimensional Sanger fingerprints of pancreatic and T, RNase digests of EPl and EP2 RNA (not shown) were compared with those of the mature glycine and threonine tRNAs as well as that of the precursor. The oligonucleotides from these digests were studied by enzymatic and alkaline digestion as described above for the sequential analysis of EP3 RNA. It was found that EP2 RNA contains the complete primary sequence of mature tRNA,Tp",c, including the 5'-terminal pG-C-sequence. The only structural difference observed was that the 3'-terminal sequence C-A-C-C-A,, in threonine    Tables I and V were  analyzed  by standard  alkaline  and   tRNA was replaced by the sequence C-A-C-C-A-C-U(-U,,,,) in EP2 RNA. Thus, the 3'-terminal heterogeneity found in the precursor molecule is preserved in this RNase P generated fragment.
A similar comparison between tRNAi$:, and EPl (Tables I and V), suggesting that the arrangement of these three segments in the precursor is 5'.EPZEPl-EP2-3'. Thus, the tRNA sequence in the precursor has the order 5'-tRNAg:y,S,,,-tRNA12;1,c-3', with precursor-specific nucleotides distributed at both termini of the molecule as well as in the inter-tRNA spacer region (see Fig. 7~).

Partial
Enzymatic Digestion Products and Complete Sequence of tRNA';'y-tRNA'r" Precursor-In order to determine the total sequence of the precursor unambiguously, it was necessary to isolate partial enzymatic digestion products with sequences that overlap the tRNA and precursor-specific regions. This was achieved by limited digestion of the precursor RNA with T, RNase or modified pancreatic RNase (t-carboxymethyl lysine-41), followed by fractionation of the products by homochromatography (22). The partial digestion products were analyzed by complete digestions with T, or pancreatic RNases, followed by standard two-dimensional fingerprinting techniques.
The end products were characterized by their position on the fingerprints and by alkaline or enzymatic digestion where appropriate.
The vast majority of these partials were derived from the tRNA sequences in the precursor molecule, and their sequences are consistent with the known structures oi [he mature tRNAs. However, four partial products which overlap the precursor-specific and tRNA regions 5550 were isolated. The deduction of their structures from the end products is illustrated in Table VII. The sequences of partial fragments Tl, CMPl, and CMP2 were deduced by making use of the known tRNA sequences in the precursor.
Knowledge of the sequences of EP3 RNA, tRNAz,&,, and tRNA"?' ACIr,C together with the overlap data establishes a unique sequence for the tRNA';'Y-tRNAThr precursor as shown in Fig.  6. The final sequence of the precursor molecule is presented in Fig. 7c with the tRNA regions arranged in the cloverleaf pattern. The arrows indicate the cleavage sites for the precursor processing enzyme, RNase P, which cleaves specifically Each product was analyzed by alkaline hydrolysis and venom phosphodiesterase digestion without dephosphorylation as described in Table   II. Yields were estimated in a scintillation counter and were expressed relative to 1 mol of the 3'.terminal or trinucleotides. at two locations to generate the mature 5' ends of the tRNAs. The precursor molecule consists of 170 nucleotides; 151 are faithful copies of the mature glycine and threonine tRNA sequences, while 19 are precursor-specific. Six precursorspecific nucleotides occur in the inter-tRNA spacer region, with ten on the 5' terminus, and the remaining three on the 3' terminus of the precursor molecule.
Nucleoticle Modifications-All T,-RNase fragments from a fingerprint of the [32P]tRNAG'y-tRNAThr precursor were digested to mononucleotides with a combination of pancreatic RNase and spleen phosphodiesterase.
The digestion products from each fragment were then fractionated by paper electrophoresis at pH 3.5, eluted, and characterized by descending paper chromatography using Systems a and b of Barrel1 (22). A total of 11 modified nucleotides were found, which agrees with the previous finding that there are four modified nucleotides in tRNA::y,& (29,30) and seven in tRNAf;",;,,, Dp in fragment t8, m7Gp in fragment t10, *mtGAp in fragment t20, Np in fragment t24u, and both Np and Ap in fragment t24 were revealed by the screening procedure noted above. T,-RNase fragment t15 has the sequence D-D-G-(residues 107 to 109). Analysis of the nucleotide composition of this fragment by descending paper chromatography using Systems a and b indicated that the Oligonucleotides t26 and t27 (Fig. 4) were partially digested with venom phosphodiesterase as described under "Experimental Procedures" and the products were f'ractionated by ionophoresis on DEAE-paper at pH 3.5. Each product was characterized by pancreatic and U,-RNase digestions.
The compositions of these products were in turn determined by alkaline and venom phosphodiesterase digestions as in Table III. VaniX.0 phosphodiesterase partial digestion productsa Enzymatic digestion products The oligonucleotides derived from pancreatic RNase digests of [32P]tRNA~1Y-tRNAThT precursor are numbered as in Fig. 5. Molar yields, determined as described in Table I,  1 I modification to form Dp was incomplete; Dp was present in molar yield (relative to Gp) higher than 1, while less than 1 molar equivalent of unmodified Up was present. Apparently, some modification had occurred at both residues 107 and 108. Oligonucleotide t16 has the sequence T-$-C-G-and occurs twice in the precursor at positions 63 to 66 and 145 to 148. Similar quantitative and qualitative analyses of the nucleotide content of this fragment indicated that these residues were modified nearly to completion, as suggested by the presence of only traces of IJp in this fragment. When amplified and labeled by the prophage induction method, the precurzor was consistently modified to varying degrees. The level of Ap modification was usually 20 to 50%; Np modification varied from only trace amounts to about 20% completed. Modifications at other positions were generally 70 to 100% completed. DISCUSSION The primary sequence of a precursor RNA to Escherichia coli tRNA;z,&, and tRNA:,[:r, ,(. has been determined. This precursor, 170 nucleotides in length, contains the complete sequences of mature glycine and threonine tRNAs, including the 3'.terminal -C-C-A triplets, along with 19 additional nucleotides located at the 5' and 3' ends of the molecule as well as between the tRNA sequences. Fig. 7c shows the sequence of the precursor with the tRNA portions arranged in the cloverleaf pattern. Eight additional base pairs could be made between the central single-stranded region and the 5'-and 3'-terminal segments, to give a more stable secondary structure according to the rules of Tinoco et al. (34). They are (positioned from the 5' end as indicated in Fig. 6) G-G-A (7 to 9) with U-C-C (82 to 84), A-A-G (85 to 87) with C-U-U (168 to 170), and G-U (90 and 91) with A-C (164 and 165). Similarly, in the case of a precursor to E. coli tRNA;'$,i,, two additional base pairs can be formed to extend the double-stranded -CCA stem to nine base pairs (2). The exact secondary structure of these precursor RNAs is still unknown, however.
When incubated with a cell-free extract prepared from sonicated E. coli cells, the precursor is converted to mature glycine and threonine tRNAs. Purified RNase P, as would be expected from the known mode of action of this enzyme (6), cleaves the molecule specifically at two sites (as indicated in Fig. 7c by the arrows), to generate the mature 5' end sequences of both tRNAs. Since it has been shown that the nuclease that processes the 3' end of tRNA precursors is different from RNase P (6,33), the presence of extra 3'.nucleotides in precursor RNA isolated from an RNase P mutant is somewhat surprising.
We have observed the presence of the 3'.terminal extra nucleotides even after prolonged labeling for 2 hours at nonpermissive temperatures.
It seems clear that maturation at the 5' end is a prerequisite for complete processing of the 3' end. Conceivably, the presence of the extra segment on the 5' terminus could render the few extra nucleotides at the 3' end inaccessible to the maturation enzyme(s), perhaps by permitting extra base pairs to form as described above.
Under the experimental conditions used, nucleotides found modified in mature glycine and threonine tRNAs are also modified in the precursor molecule. Although the modification at some residues is incomplete, it is clear that modification enzymes are capable of using the precursor RNA as their substrate.
The observed undermodification might reflect a lower rate of enzymatic modification for the precursor due to secondary or tertiary structural differences between mature tRNAs and their precursors, as suggested by Schaefer et al. (35).
The terminal-CCA segments of both tRNAi:Y,i, and tRNA$;,,c are internally located in the precursor RNA sequence. Therefore these -CCA sequences appear to be transcribed directly from the DNA, as in the case of the precursor to tRNA7,y,',l (2). On the other hand, Barrel1 et al. (4) have shown that a tRNAs"'-tRNA""' precursor specified by bacteriophage T, lacks the CCA sequences for both tRNAs. In the latter case, the enzyme, tRNA nucleotidyltransferase, is probably responsible for the addition of the terminal CCA sequences (36). In another di-tRNA precursor (tRNA"'"-tRNA',eU) specified by bacteriophage T,, the tRNA';"' at the 5' end of the precursor lacks the CCA, while the tRNA',"" sequence at the 3' end of the precursor ends normally in -CCA.2At the moment it is not clear what significance, if any, can be attached to the presence or absence of the CCA sequence in tRNA precursor molecules.
Genetic and biochemical studies have shown that three tRNA genes (glyT   thrT  tyrT) are contained on the small section of the E. coli chromosome which is incorporated onto "C. Guthrie, private communication.  Table II. The yields of the products  were estimated  by liquid scintillation  counting  and were  expressed  relative 6. Overlaps between the primary sequences of tRNA$,i, and tRNAX&,c, the sequences of partial digestion products, and the sequence of the 5'-terminal segment, EP3 RNA. Sequences of partial digestion products were deduced as summarized in Table VII. The sequence of EP3 RNA (residues 1 to 10) was determined as indicated in the text. Precursor-specific nucleotides are indicated by a line above the sequences.   This gene order is now uncertain, however, since a tRNA"lY 2-tRNA"'"' precursor RNA specified by this same phage has been isolated and sequenced.
Although the sequencing data has shown that glyT and thrT are adjacent on the E. coli chromosome and are transcribed into a single polynucleotide chain, the relative location of tyrT has not been established. Equimolar synthesis of these three tRNAs in induced wild type E. coli cells lysogenic for the defective phage Xh80dglyTsuA36 thrT tyrT has been previously demonstrated (17). Under the conditions used in our labeling experiments with RNase P mutant cells, tRNA',':':ll",c is also amplified in amounts equal to tRNAg$,2, and tRNA$;,,c. This RNA migrates on the gel with the electrophoretic mobility expected for mature tyrosine tRNA (banal A, Fig. l), and gives a T, RNase fingerprint characteristic of completely processed tRNATY:, :,. (32). It is possible that the initial transcript contains all three tRNAs in tandem and the tRNA:y,,:, is rapidly removed, even at the nonpermissive temperature. Thus, enzymes other than RNase P might be involved in the post-transcriptional processing of multi-tRNA precursors. Such nuclease activities have been observed by Schedl (33), and by Ghysen and Celis (37). Since the tRNA"lY '-tRNAT"' precursor lacks a 5'.terminal nucleoside triphosphate, this molecule seems to be a partially processed intermediate generated by nucleolytic cleavage(s) from a still longer primary transcript.
Joint transcription of tRNA genes has been observed for six of the eight bacteriophage T4-coded tRNAs (3). Recently, evidence has been presented indicating that two tandem tRNA',?<,,-',,. genes in E. coli are also transcribed together (37). In addition, a large precursor containing several copies of a tRNA:i:l; ",(. sequence, and a precursor containing multiple tRNA::;r,l sequences have been identified in E. coli strain A49 (Ref. 15).3 Thus, biosynthesis of tRNA via multi-tRNA precursors seems to be a common feature of bacterial tRNA gene transcription.