Nudeotide sequences of two serine tRNAs with a GGA anticodon: the structure-function relationships in the serine family of E. eoti tRNAs

We have determined the nucleotide sequence of the major species of E.coli tRKA and of a minor species having the same GGA anticodon. These two tRNAs should recognize the UCC and UCU codons, the most widely used codons for serine in the highly expressed genes of E.coli. The two sequences differ in only one position of the D-loop. Neither tRNA has a modified adenosine in the position 3'-adjacent to the anticodon. This can be rationalized on the basis of a structural constraint in the anticodon stem and may be related to optimization of the codon-anticodon interaction. Conparison of all E.coli serine tRNAs (and that encoded by bacteriophage T4) reveals characteristic (possibly functional) features. Evolutionary analysis suggests an eubacterial origin of the T4tRNA^ gene and the existences of a recent common ancestor for the tRNAger a n d tRNASer g e n e s. INTRODUCTION The degeneracy of the genetic code is such that most of the twenty anlno acids are specified by more than one code word. In particular, serine is encoded by six codons arranged in two blocks that differ in the first and the second codon, i.e., the quartet UCN (where N C,U,G or A) and the doublet (AGY where Y C or U). In order to read these six codons during mRNA decoding, tRNAs with different anticodons are required. In mitochondria, only two tRNA lsoacceptors are needed, since one having a UGA anticodon reads the entire family of UCN codons and another tRNA with a GCU anticodon reads the two AGY codons. In the eukaryotic .or prokaryotic cytoplasm a minimum of four lsoacceptors are needed. The analysis of more than 33 tRNAfl from different origins shows that there are mainly five anticodon types: IGA, U*GA, (U* is modified), GGA, CGA, and GCU. In addition to these lsoacceptors (having different anticodons), other lsoacceptor species have been found which differ in only the modified nucleoside levels (isocodlng species) (for reviews see refs 1-2). In E.coli three types of tRNA^ were sequenced; they had the anticodon U*GA, GCU and CGA (3). The object of this article is to present the sequence of the fourth type of E.coli serine tRNA (GGA anticodon) which © IRL Press Limited, Oxford, England. 5697 Nucleic Acids Research reads UCC and UCU codons. Some time ago, one of us (D.S.) reported the identification and the purification of five serine tRNAs from E.coli K12. A major species (called Ser 1) and a minor (Ser V) exhibited a coding response to poly UC and were taken to have the GGA anticodon (4). Subsequently, these two tRNAs were shown to form Gly anticodon-anticodon complexes with E.coli tRNAy*cc and yeast Arg — tRNA[j*cu (5-6). We report here the complete primary structure of these two tRNAs. Since these sequences complete the structural work on the serine tRNA family of E.coli, it is now possible to consider the hypothesis concerning the role of these tRNAs in regulation and mENA decoding. MATERIALS AND METHODS The two tRNA|er (corresponding to tRNA?er atui tRNA ) WjA I V were isolated from E.coli K12, strain CA 24A as previously published (4). Due to the inconsistencies of nomenclature in the serine tRNAs (see Table 1), we will distinguish them by their anticodon sequence. The tRNAs have been maintained at -80°C, since that time. 3'-labelling of 1 pg samples was performed by the Incubation of tRNA with [P]pCp (3000 Ci/aaole) and T4-RNA ligase (from P.L. Biochemlcals) as described in (7). Purification followed on 0.08 cm thick, SO cm long gels aade of 15Z polyacrylamide with 50 mM Tris-borlc acid buffer at pH 8.3 containing 7M urea and 10 mM EDTA. Both tRNA samples gave a single radioactive band, and their sequences were deteroined by the now standard rapid gel sequencing techniques (8)• The modified nucleosides were determined by a variation of the Gupta and Randerath method (9). A sample of 5 pg of each tRNA was partially hydrolyzed in 5 Pi of deionized formamide at 100°C for 4 nin. Chromatographic analysis was done by the separation of nucleoside diphosphates on PEI-cellulose thin layer plates in 0.55 M ammonium sulfate at 4°C or by the separation of nucleoside monophosphates (produced by an additional digestion by PI nuclease) on two-dimensional cellullose thin layer plates. RESDLTS AND DISCUSSION The serine tRNA^G^ sequences and their analysis The two serine tRNAa are composed of 88 nucleotldes, 16 of which are located in the long extra ana (Fig.la). These tRNAs along with the E.coli tRNA^" have the shortest extra arms among all sequenced serine tRNAs (3). The two tRNAs differ by one nucleotide; there is a C in position 20 of