The corrected nucleotide sequence of yeast leucine transfer ribonucleic acid

BIOCHEMICAL vol. 51, No. 4, 1973 AND BIOPHYSICAL RESEARCH COMMUNICATIONS THE CORRECTED NUCLEOTIDE SEQUENCE OF YEAST LEUCINE TRANSFER RIBONL'CLEIC ACID Simon H. Chang, Su Kuo, Erin Hawkins Department of Biochemistry, Louisiana Baton Rouge, Louisiana Received March and Nancy R. Miller State University, 70803 1,1973 SUMMARY The nucleotide sequence of "Renaturable" leucine transfer RNA from Baker's yeast has been re-investigated. The results showed that (i) this tRNA has a sequence of DCD at positions 19-21, (ii) it has an anticodon m5CAA and (iii) it has a pseudouridine at position 40. The nucleotide (tRNALFU) There 19-U, (1) has been reported however, three are, quences, sequence namely (ii) (i) the tRNA has been (2) independently leucine of discrepancy between sequence of DDCl (1) or DCD (2) CAA (1) or m5CAA (2) at position re-analyzed 40. The results are RNA (1, these 2). two se- at positions and (iii) The nucleotide carefully. transfer by two laboratories lines the anticodon or a cytidine of "Renaturable" a pseudouridine sequence of this reported in this communication. MATERIALS Baker's tRNAL$ purified ribonuclease nucleotides tRNA was prepared as previously (Sankyo). in a DEAE-cellulose previously yeast (2). were AND METHODS described The products (Whatman according DE-23) (4) were column Fractions under peaks combined, and the urea to Holley was degraded separated with with (3). T, by chromatography the conditions described containing the questioned oligo- removed by gel filtration in 1 The abbreviations used are: D, 5,6-dihydrouridine; Y, pseudouridine; m5C, 5-methylcytidine; ac"C, @-acetylcytidine; Cm, 2'-Omethylguanosine; m'%, N2-methylguanosine; m$G, N2,N2-dimethylguanosine; Pan RNase, pancreatic ribonuclease. One A,o unit is defined as that amount of material per ml of solution which produces an absorbance of 1 in 1 cm light path at 260 nm. Copyright AN rights 0 I9 73 by Academic Press, Inc. of reproduction in any form reserved. 951 Vol. 51, No. 4,1973 BIOCHEMICAL AND BIOPHYSICAL columns of Biogel P-2 (Bio Pad). further separated by, first, Oligonucleotides RESEARCH COMMUNICATIONS under each peak were incubation with bacterial alkaline (Worthington BAPF) and then chromatography in isobutyric (66:1:33, solvent A) or by high voltage electrophoresis nucleotide composition of each oligonucleotide with pancreatic ribonuclease (Worthington) phosphatase acid-NQOH-Hfl in 7$ HCOOH. The was analyzed by digestion and bacterial alkaline phos- phatase, followed by chromatography of the products in isopropanolWOH-PI,?0 (7:1:2, solvent B). The 5’ terminal nucleoside of each oligo- nucelotide was determined by degradation of the 3’ dephosphorylated oligonucleotide with snake venom phosphodiesterase (Worthington) separation of the products in solvent B. three questioned oligonucleotides The oligonucleotide The nucleotide and sequences of all were determined with a commonprocedure. was incubated with polynucleotide phosphorylase (P-L Biochemicals) in the presence of inorganic phosphate and bacterial phosphatase. A trinucleotide alkaline from the 5’ end was isolated by chromatography of the products in solvent A or n-propanol-WOH-H-4 bation with bacterial vent A. The trinucleotide solvent C). (55:10:35, alkaline This purified pancreatic purified by incu- phosphatase and re-chromatography in sol- trinucleotide was analyzed by degradation with ribonuclease and chromatography of the products in solvent B. Nucleosides or nucleotides were identified at neutral, was further acidic, and alkaline by their ultraviolet spectra pH's. RESULTS Figure 1 shows the chromatographic pattern of the products ob- tained by degradation of the tRNA with T1 RNase. Peak 14 containing pentanucleotides CYCAGand UAUCG,peak 16 containing two the hexanucleotide DCDAAGand peak 17, the anticodon fragment were analyzed with the procedure described above. Table I. The results of these analyses are summarized in Based on these results and those we reported previously 952 (2) a BIOCHEMICAL Vol. 51, No. 4,1973 2.0 I AND BIOPHYSICAL RESEARCH COMMUNICATIONS 4 16 6 E c 0 21.0 a 2 I 0.0 r' : II FRACTION NUMBER Fig. 1. Chromatography of T1 RNasedigest of tRNALTu-. 315 A2t;o UnitS of tRNAL5Uwere incubated with 1000 units of T1 RNaseat 3’7’ for 10 hours. The mixture was then applied on a column (1 x 60 cm) of DEAEcellulose. The column was eluted with a linear gradient (0 to 0.4 M) of NaCl in 7 M urea buffered with 0.02 M Tris-HCl, pH 7.5. Total volume of the gradient was 2 liters. Fractions of 3 ml were collected every 10 min. AOH C k PG-C G-C U-A U-A G-C U-G AA GmcGAG G D G UJC CD AA Y U c m’G m=cA A Fig. 2. The corrected leaf form. nucleotide sequence of yeast tRNAL5U in the clover- 953 TABLE Nucleotide sequences Peak No. * of three fragments Products from Pan mase + alkaline phosphatase degradation obtained I by degradation of tRNAL5" Nucleoside from snake venom phosphodiesterase depradation with T1 ribonuclease. 5' trinucleotide from polynucleotide phosphorylase degradation Nucleotide sequence 12" 2C, $2 AG C ck (c)* C$CAG 14 2D, C, AAG D DCD (D)% DCDAAG 15 U, m5C, A$, AAmlG A A$U (U)"" A@JmSCAAmlG The slower M Nucleoside moving obtained band from high by degradation voltage of electrophoresis the trinucleotide in 7% HCOOH. with Pan RNase. Vol. 5 1, No. 4,1973 corrected its nucleotide clover-leaf BlOCHEMfCAL AND BIOPHYSICAL sequence yeast secondary for RESEARCH COMMUNICATIONS tRNAL~U is shown in Fig. 2 in structure. DISCUSSION The three tRNALy codon (ii) has a sequence m5CAA and (iii) are (iii) the described in tRNA structure techniques represent (1, It with are a similar findings this correct the noting that number plus of yeast the tri- charges. can be achieved and re-chromatography nucleotides so purified provide clear solvent cut and the rest of two different diphosphates The removal in (2) 2 must of oligonucleotides isolated since of these by dephosphorylation phosphatase (i) The cor- or tetranucleotides 5’ and shown in Fig. the degradation nucleoside of negative that from Leu tRNA 3 . (i) (1). the fact results 2 are has an anti40. and coworkers sequence in it previously final phosphorylase, with (ii) we reported upon by the that I and Fig. at position of Kowalski structure is worth 19-U, communication contaminated diphosphates results is agreed polynucleotide usually the 2) indicate the shown in Table has a pseudouridine with with points of DCD at positions it in agreement agrees rections this important with A (5). all nucleoside bacterial Tri- information have alkaline or tetra- for sequence analysis. ACKNOWLEDGMENTS The authors wish to thank Drs. S. Kowalski, J. Chirikjian and J. R. Fresco in Princeton University for their suggestive discussion on the sequence of this tRNA. This work was supported in part by Grant No. GB 17124 from the National Science Foundation. REFERENCES 1. Kowalski, S., 2. Chang, S. H., 265 0~~1). 3. Halley, 4. Chang, Letters, 5. RajBhandary, R. W., Yamane, 'I. Miller, N. R. and Harmon, Biochem. S. H., Harmon, 11, 81 (190). U. L., and Fresco, Biophys. -Res. J. R., Commun ., lo, communication. 955 z, 385 (1971). C. W., w-2 FEBS Letters C. W., Munninger, Personal Science, K. and Miller, Q', 186 (1963). N. R., FEBS