Total Synthesis of a Tyrosine Suppressor tRNA Gene

The chemically synthesized gene for Escherichia coli tyrosine suppressor tRNA has been joined to both plasmid (ColE1 ampr) and bacteriophage (Charon 3A) vector chromosomes after the latter had been digested with the restriction endonuclease EcoRI. Suppression of both bacterial (trpA, his, lacZ) and bacteriophage lambda amber mutations (Aam32, Bam1) has been demonstrated after transformation of E. coli with the recombinant DNA molecules carrying the synthetic suppressor tRNA gene. The cloned synthetic gene has been reisolated from the vector chromosomes after digestion of the latter with EcoRI restriction endonuclease and characterized in regard to its size and its ability to serve as a source of suppressor activity in further transformation experiments. This synthetic gene has also been shown to suppress bacterial amber mutations after it had been incorporated into the E. coli chromosome as part of a lambda prophage. Transcription, in vitro, of the cloned synthetic suppressor gene gave a product which, on treatment with a crude E. coli extract, afforded the tyrosine suppressor tRNA precursor. The latter was characterized by two-dimensional fingerprinting after digestion with T1-RNase. Exposure of the in vitro transcript to RNase P Selectively released the 41-nucleotide-long fragment characteristic of the 5'-end of the tRNA precursor. Thus, the nucleotide sequence of the cloned gene is accurate and its expression is controlled by its promoter.

By use of polynucleotide kinase and polynucleotide ligase, the 10 deoxyoligonucleotide segments, whose syntheses have been described in accompanying papers, have been joined to form the 62-nucleotide-long DNA corresponding to the promoter region of an Es&erichia coli suppressor tRNA gene. The following sequence in the joining reactions was used to obtain error-free and optimal yields of the products: 1) joining of Segment P-l to P-3 in the presence of Segment P-2; 2) joining of Segments P-4 to P-7 to form Duplex Ip4-,]; 3) joining of Segments P-8 to P-10 to Duplex [Pd-,] to form Duplex [P4-10]; and finally, 4) joining of P-(1 + 3) and P-2 to Duplex [P4-10] to form the total promoter Duplex [P].
As a requirement for the total synthesis of the Escherichia coli tyrosine suppressor tRNA gene, the nucleotide sequence in its promoter region was determined (2,3). In further work, a plan was formulated for the synthesis of this part of the gene (1,4). This plan, which is shown in Fig. 1, included the first 51 nucleotides of the promoter sequence as well as the EcoRI endonuclease-specific sequence at the appropriate terminus. Thus, chemical syntheses of 10 deoxyoligonucleotide segments (P-l to P-10) were required, and the preceding two papers (1,4) have documented the successful conclusion of this phase of the work. The present paper reports on a systematic study of Paper 14 in this present series, is Ref. 1 the polynucleotide ligase-catalyzed joining of the chemically synthesized segments; this has led to the successful synthesis of the total promoter duplex.
The plan shown in Fig. 1 for segmentation of the promoter duplex emerged as a compromise between the demands on chemical synthesis and possible errors or difficulties in enzymatic joinings. The palindromic features in the promoter structure (2) were anticipated to cause some difficulties in error-free joinings. Thus, Segment P-5 has a great deal of selfcomplementarity (Fig. 2). Similarly, there is considerable complementarity between Segments P-7 and P-10 as shown in Fig. 3. Self-complementarity to varying degrees within individual synthetic segments was found to be unavoidable on several occasions during previous work (e.g. 5-7), and reasonably satisfactory solutions were found in all cases. The most serious aspect in the present work seemed to be the homology at the 5'-end between Segments P-2 and P-5. Therefore, they could substitute for each other and, as a result, in joining reactions in which Segments P-2 to P-4 ( Fig. 4A) or Segments P-3 to P-5 ( Fig. 4B) are used, duplexes longer than the expected products could form. This was in fact found to be the case (see below). Therefore, it was clear that, in the planning of the enzymatic work, Segment P-2 could only be grouped with Segments P-l and P-3 and that Segment P-5 could be grouped with Segments P-4, P-6, and additional segments beginning with P-7.
In initial studies on grouping of the segments for joining purposes, the joining of Segment P-l to Segment P-3 in the presence of Segment P-2 proceeded well (3). Similarly, Segment P-5 joined well to Segment P-7 in the presence of Segment P-6. Further, although Segments P-4 and P-6 did not join significantly in the presence of Segment P-5,' the four-component system consisting of Segments P-4 to P-7 gave an essentially quantitative yield of the expected duplex. In further work, the joining of Segment P-8 to Segment P-10 with Segment P-9 as the template was found not to proceed satisfactorily.' In this three-component system, no advantage accrued when a fourth component, Segment P-7, was added, presumably due to the complementarity between P-7 and P-10 ( Fig. 3). In another attempt, one-step joining of the seven segments (P-4 to P-10) was investigated. However, the extent of total joinings was unexpectedly low. From these studies, it became clear that the joining of Segments P-4 to P-10 must be carried out in two steps. The first step would constitute the formation of Duplex [P,-71. The resulting duplex should then be extended by the addition of Segments P-8 to P-10.
I Unsatisfactory joinings using three-component systems have been observed in previous work (6,7). This is, presumably, because one or more of the segments can adopt alternative bihelical structures.
--r m Y) --Synthesis of Tyrosine Suppressor tRNA Gene of undesired duplexes longer than expected from Segments P-2 to P-4 (A) or from Segments P-3 to P-5 (El). Additional joinings in both cases result from the homology between Segments P-2 and P-5.
FIG. 5. Plan adopted for the stepwise joining of the chemically synthesized Segments P-l to P-10 to form the total DNA corresponding to the promoter.
Thus, in Step 2, Segments P-l and P-3 were joined.
In Step 2, Segments P-4 to P-7 were joined to form the corresponding duplex. In Step 3, the duplex from Step 2 was extended by joining in Segments P-8 to P-10. Finally (Step 4), Segments P-(1 + 3) and P-2 were joined to Duplex [P,.,,,]. to this column, the latter was washed with Buffer A (6 ml) and the enzyme was then eluted with 6 ml of Buffer A containing 0.6 M KCl. This eluate was dialyzed against Buffer A + 50% glycerol and stored at -20°C at a concentration of 8,000 units/ml (9). Recovery of ligase activity from these procedures has varied between 30% and 50%.

RESULTS
Joining Experiments using (a) Segments P-2 to P-4 and (b) Segments P-3 to P-~-AS shown in Fig. 6a, the combination of Segments P-2, P-3, and P-4 gave a product containing four segments as the major product. Formation of this product would be expected to occur as in Fig. 4A. The system containing Segments P-3, P-4, and P-5 gave several products (Fig.   6b) which contained two, four, six, seven, and eight segments. in the mixture were as described previously (5). Before the addition of ATP, enzyme, and dithiothreitol, the segments were heated at 97°C for 2 min and then cooled to 18°C during 15 min. excess and the desired product was purified after the joining reaction. Unphosphorylated P-l (8.5 nmol of about 50% pure sample) and ["P](P-3) (4.5 nmol) were joined using T4-ligase in the presence of ["'P](P-2) (4.9 nmol) as the template. As shown in Fig. 7, the product, P-(1 + 3), with a tailing impurity of somewhat shorter size was obtained in a yield of 53% as based on P-3. Pure P-( 1 + 3) was obtained by preparative gel electrophoresis in an amount of 3 nmol from two reactions carried out on the scale described above. Characterization of P-(1 + 3) thus obtained is given in Table Ia. Thus, when the product was digested to 3'-and 5'-mononucleotides, the radioactivity was found only in dCp and pdG, respectively.
Preparation of Duplex (P4-J Containing Segments P-4 to P-7-Formation of the self-complementary duplexed structure from Segment P-5 (Fig. 2), appeared to be concentration-dependent and, therefore, it was desirable to keep the concentration of this segment at about 5 pM or less. There are analogies for this from previous experience (5) and from the present work; at 40 pM concentration of Segment P-5, the reaction mixture containing the above four segments did not give any joining.
However, when a 5-nmol scale reaction was performed using the conditions shown in Fig. 8, the joining reaction occurred as shown, and 75% of the total radioactivity originally present was converted to the expected duplex. The result indicates a 100% utilization of Segment P-4, which was present in a limiting amount, in the formation of Duplex [P,-,I. In repetitions of the reaction at the same scale, yields of around 85% of the duplex were obtained. The duplex was isolated by gel filtration through a Sephadex G-100 column (1.2 x 90 cm) (Fig. 9).
Characterization of Duplex [P4-i] is given in Table Ic. When the duplex was first treated with bacterial alkaline phosphaable until recently, has now been purified by high pressure liquid chromatography.
As expected, it joins to Segment 3 in high yield in the experiment of Fig. 7.  . The mixture was heated at 97°C for 2 min and then cooled slowly (15 min) to 18"C, ATP (final concentration 100 PM), dithiothreitol (10 mM) and T4-ligate (400 units/ml) were added at 4°C. The reaction was carried out at 5°C. Kinetics of the joining were followed by electrophoresis on a 15% polyacrylamide gel using 0.5-d aliquots from the mixture. Gel hands were cut out and radioactivity was determined by measuring Cerenkov radiation. XC, xylene cyanol; BPB, bromphenol blue.

Synthesis of Tyrosine Suppressor tRNA Gene
tase and then digested to 3'-mononucleotides, radioactivity, besides being in inorganic phosphate, was found only in dCp. When the duplex was digested to 5'-mononucleotides, the radioactivity was found in pdG and pdT in the expected ratio of 1:3.
Synthesis of Duplex [P.4.1~] by Addition of Segments P-8, P-9, and P-10 to Preformed Duplex (P,-J--For effective joining of the Segments P-8 to P-10 to the preformed Duplex [P4-i], Segments P-8 and P-9 were used in excess (about 2 molar equivalents) over the latter. Furthermore, Segment P-10 was added in even larger excess (about 4.5 molar equivalents) since the joining of the latter to Duplex [P.+-q] was found to be very slow. The synthesis of Duplex [P,-N] was carried out as described in Fig. 10 and the kinetic data of joining are shown. After 1 h the only new product detected was Duplex [P,-,I. Since no [P,_,] was detected, the joining of P-8 and P-Synthesis of Tyrosine Suppressor tRNA Gene 5785 9 probably occurred concomitantly. The joining of P-10 was rate-limiting, but after 24 h, 60% of the starting duplex [P,-,] had been converted to [P,-,"I and another 23% was present as [Pde9]. Pure [P4-10] was obtained by preparative electrophoresis on a 15% polyacrylamide gel. In more recent experiments, the conversion of Duplex [P,-,] to [P4-9] + [P4-Io] has been higher than that recorded above, only a trace of unreacted [PdeT] being observed.
Characterization of Duplex [P,-,"I by degradation to 3'-and 5'-mononucleotides is given in Table Id. Thus, when [P~-M] was digested to 3'-mononucleotides, following bacterial alkaline phosphatase treatment, radioactivity was found in dCp and dTp in the expected ratio of 4:l. A higher molar ratio observed for inorganic phosphate was caused by some dephosphorylation of mononucleotides by a phosphatase which contaminated the spleen phosphodiesterase preparation used. Degradation of [P4-,"] to 5'-mononucleotides gave pdA, pdG, and pdT in the expected ratio of 1:1:4. Similar degradations of [P,-e] gave distribution of radioactivity in the expected mononucleotides in correct ratios.
The Total Promoter Duplex [PJ, Containing Segments P-1 to P-lo-The 5'-end of Segment P-l, which later is to be joined to the structural gene (following paper), should carry a [""PIphosphate group of high specific activity, while the opposite terminus, which contains the self-complementary . The ligase reaction mixture described in Fig. 8 was applied to a column of Sephadex G-100 (1.2 x 90 cm) at 4°C. Elution was performed using 50 mu triethylammonium bicarbonate (pH 7.5) as the eluant. One-half-milliliter fractions were collected every 10 min.
EcoRI endonuclease sequence, should not carry a phosphate group during any of the ligase reactions. Therefore, the singlestranded polynucleotide P-(1 + 3) as prepared above (Fig. 7) was phosphorylated using [y-"P]ATP which had specific activity 65 times higher than that of the ATP which had been used for phosphorylation for the purpose of joinings at the internal sites. Phosphorylation (kinetics not shown) was complete under the standard conditions and the radioactivity, on degradation of the product to 5'-nucleotides, was found only in dpG (Table Ib).
As is seen in Fig. 11, about 40% of [P4-10] was converted to the promoter, [P]. Joining of [P4-10] to P-(1 + 3) also occurred in the absence of P-2 to give, as expected, slightly shorter product (Fig. lle). From a 550-pmol scale reaction of [P4-10], 169 pmol of the duplex [P] was obtained as a final product.
Characterization of the isolated [P] is given in Table Ie. Thus, when Duplex [P] was first treated with bacterial alkaline phosphatase and then digested to 3'-mononucleotides, radioactivity was found in dCp, dAp, and dTp in the expected ratio of 5:2:1. Radioactive inorganic phosphate, which was produced from the 5'-dG end of [PI, had the expected high specific activity and was recovered in the correct molar amount. Degradation of [P] to 5'-mononucleotides gave radioactive pdA, pdG, and pdT in a ratio of 1:3:5, which again was as expected.
Comments-The enzymatic joining of chemically synthesized deoxyribo-oligonucleotide segments still requires considerable empirical work in order to arrive at the optimal strategy for the synthesis of the final product. Economy in chemical synthesis will continue to be the more important aspect in determining the segments to be synthesized for two reasons: First, chemical synthesis still constitutes the more time-consuming and laborious part of the work; and secondly, because of the considerable choice in segment combination in joining reactions, reasonably satisfactory schemes for error-free joinings can be worked out. Once the duplexes with the comple-  in amounts to those already present) were added. For analysis, gel (550 pmol) and [ "P](P-2) (1780 pmol) were added. The final reaction bands were cut out and the radioactivity was determined by Cerenkov radiation. Kinetics obtained are shown. Channel e shows the experimixture (121 ~1) contained other components as described under ment in which the reaction mixture containing Duplex [Pd.l,,] and "Materials and Methods," including 83 pM ATP and 400 units/ml of Segment P-( 1 + 3), as described above, was incubated at 5°C for 6.5 T4-ligase. The reaction was performed at 5°C. At the time intervals h in the absence of Segment P-2.
mentary protruding ends have been prepared, their end-toend joining to form larger duplexes, and, finally,. the total duplex proceeds rapidly and without difficulty. This has been uniformly the case in the work reported previously (10, 11) and that described in the following paper (12).